Non-typeable haemophilus influenzae vaccines and their uses

ABSTRACT

Provided are non-typeable  Haemophilus influenzae  vaccines useful for the treatment of chronic obstructive pulmonary disease and asthma in a patient. In certain aspects, the vaccine is a monobacterial vaccine. Bacterial strains for use in the vaccines of the present application are also provided.

This application is a National Stage under 35 U.S.C. § 371 of PCTInternational Application No. PCT/IB09/007303, filed Sep. 17, 2009,which was published in English on Mar. 25, 2010 as WO 2010/032141 underPCT Article 21(2), which claims the benefit of U.S. Provisionalapplication No. 61/097,729, filed on Sep. 17, 2008, all of which arehereby incorporate herein by reference in their entireties.

TECHNICAL FIELD

The present application relates to non-typeable Haemophilus influenzaevaccines and their uses for the treatment of pulmonary diseases such aschronic obstructive pulmonary disease and asthma. The presentapplication also relates to novel strains of Haemophilus influenzaeuseful for treating pulmonary diseases such as chronic obstructivepulmonary disease and asthma.

BACKGROUND

Chronic Obstructive Pulmonary Disease (COPD) due largely to theinhalation of tobacco smoke is a major cause of morbidity and mortalitybeing the fourth most common cause of death in the World (Lung DiseaseData (2008). American Lung Association). According to estimates by theNational Heart, Lung and Blood Institute, in 2007, the annual cost in USfor COPD was $42.6 billion, including $26.7 million in direct healthcare (Lung Disease Data (2008). American Lung Association). The failureof established medical therapy to substantially improve outcome has beendisappointing and new management strategies are needed (GlobalInitiative for COPD (2006). Global Strategy for the Diagnosis,Management & Prevention of COPD, which can be found on the web site ofthe Global Initiative for Chronic Obstructive Lung Disease). Theunderlying pathology of COPD includes a narrowing of the small airwaysand destruction of lung parenchyma, outcomes that at least in part are aconsequence of an inflammatory process. Recurrent acute exacerbationsare linked to worsening of airflow obstruction and health statuspossibly because they reflect more intense intrabronchial inflammation.The cause of this inflammatory response is complex, but in part includesbacterial colonization of the intrapulmonary bronchus mucosa which hasbeen damaged by inhalation of toxic material (Sethi, 2006, Chest129:223-224).

Acute exacerbations are critical events in the natural history of COPDand are more common in severe disease. Acute exacerbations reducequality of life, accelerate further decline in lung function, and aremajor determinants of hospitalization and death (Global Initiative forCOPD, 2006; Niewoehner, 2006, Am. J. Med. 119(10 Suppl. 1):38-45;Wedzicha & Seemungal, 2007, Lancet 370(9589):786-96; Sethi, 2006, Chest129:223-224; Anzueto, 2007, Clin. Chest Med. 28(3):609-16, vii; and Looket al., 2006, Proc. Am. Thorac. Soc. 3(6):482-3). Once patients arehospitalized with an exacerbation they remain at high risk of hospitalreadmission or death (Sin & Tu, 2001, Am. J. Respir. Crit. Care Med.164(4):580-4).

Prevention of COPD exacerbations is a major goal of management (GlobalInitiative for COPD Guidelines, 2002; Celli & MacNee, 2004, Eur. Respir.J. 23(6):932-46; O'Donnell et al., 2007, Can. Respir J. 14 Suppl.B:5B-32B), with prevention or reduction in the number of exacerbationslikely having beneficial effects on the long-term clinical course of thedisease and patients' quality of life (Burge et al., 2000, BMJ320(7245):1297-303; Calverley et al., 2003, Chest 124(4):1350-6; Vinckenet al., 2002, Eur. Respir. J. 19(2):209-16; and Casaburi et al., 2002,Eur. Respir J. 19(2):217-24).

Asthma is a chronic inflammatory condition of the airways characterizedby reversible airway obstruction, and has traditionally been classifiedas extrinsic (due to allergic reaction to inhaled allergens such aspollens and house dust mite) or intrinsic (not due to classicalallergy), the mechanism for which is unknown. Extrinsic asthma is alsoreferred to as “allergic” asthma, whereas intrinsic asthma is alsoreferred to as “non-allergic” or “idiopathic” asthma.

In contrast to COPD, asthma is a typically chronic condition involvingthe respiratory system in which the airways occasionally constrict,become inflamed, and are lined with excessive amounts of mucus, often inresponse to one or more triggers. These episodes may be triggered bysuch things as exposure to an environmental stimulant such as anallergen, environmental tobacco smoke, cold or warm air, perfume, petdander, moist air, exercise or exertion, or emotional stress. Inchildren, the most common triggers are viral illnesses such as thosethat cause the common cold. This airway narrowing causes symptoms suchas wheezing, shortness of breath, chest tightness, and coughing. Theairway constriction responds to bronchodilators. Between episodes, mostpatients feel well but can have mild symptoms and they may remain shortof breath after exercise for longer periods of time than the unaffectedindividual. The symptoms of asthma, which can range from mild to lifethreatening, can usually be controlled with a combination of drugs andenvironmental changes.

In a recently reported study based on diagnosed asthma subjects, asthmawas classified based on differences in eosinophil and neutrophil countsin sputum (Simpson et al., 2006, Respirology 11:54-61). The subjects inthe study were divided into different asthma subtypes based on thepresence of these cell types compared to healthy control subjects.Several asthma sub-types were identified including neutrophilic asthma(>61% neutrophils) and eosinophilic asthma (>1.01% eosinophils). Theneutrophilic asthma group comprised approximately 20% of the overallnumber of asthmatics. The study further reported persistent neutrophiliain the majority of these subjects over both short term (4 week) and longterm (mean 5.3 years) intervals between sampling despite no subjectreporting respiratory tract infection during the month prior toassessment. While subjects with asthma were found to have higher levelsof intracellular bacteria and macrophages than healthy controls, nosignificant differences were found between neutrophilic asthmatics andthe other asthma groups. Indeed, the levels of bacteria found werestated to be less than that consistent with acute bacterial infections,and the report concluded there was no evidence of bacterial infection toexplain the inflammatory process of neutrophilic asthma.

Bronchitis is an inflammation of the bronchi (medium-size airways) inthe lungs. Chronic bronchitis (CB) is not necessarily caused byinfection and is generally part of COPD syndrome. CB is definedclinically as a persistent cough that produces sputum (phlegm) andmucus, for at least three months in two consecutive years.

Non-typeable Haemophilus influenzae (NTHi) is the most common pathogenicbacteria associated with chronic bronchitis (CB) (Sethi and Murphy,2001, Clin. Microbiol. Rev. 14:336-363). NTHi can be found in the upperairways (e.g., nose, middle ear, throat and sinuses) of healthy patientsand patients with CB (Sethi and Murphy, 2001, Clin. Microbiol. Rev.14:336-363) as well as several locations of the respiratory tract,including the lumen, adhering to mucosal epithelial cells in theinterstitium of the submucosa (Moller et al., 1998, Am. J. Respir. Crit.Care Med. 157:950-56). Studies of non-obstructive and obstructive CBhave observed that a large proportion of patients have persistentinfection with NTHi (Murphy et al., 2004, Am. J. Respir. Crit. Care Med.170:266-72).

FIG. 4 is a side perspective view of the rim cover of FIG. 1 engagedwith the rim of a wheel, the view is of the side of the wheel adjacentthe vehicle;

Both NTHi and Staphylococcus aureus have previously been shown to inducenon IgE mediated and enhanced IgE-mediated histamine release from mastcells obtained by broncheoalveolar lavage from the airways of patientswith CB. In the case of NTHi, it has been reported that exotoxin may beresponsible for the enhancement of IgE mediated histamine release(Clementsen et al., 1990, Allergy 45: 10-17). Immune cells isolated frompatients with CB during acute exacerbations have been shown to be bothsensitized and hyperactive to the patient's own bacteria (Norn et al.,1994, Agents Actions 41, Special Conference Issue 1994:C22-C23). Severalstudies have also reported specific IgE antibodies produced in responseto respiratory infection by fungi (e.g., Aspergillus) and viruses (e.g.,respiratory syncytial virus, parainfluenza virus (Welliver et al., 1982,J. Pediatrics 101:889-96)) and bacteria (S. pneumoniae (Kjaergard etal., 1996, APMIS 104:61-67; Tee and Pepys, 1982, Clin. Allergy12:439-50; Pauwels et al., 1980, Allergy 157:665-9), S. aureus (Rhode etal., 2004, Respir. Med. 98:858-64; Tee and Pepys, 1982, Clin. Allergy12:439-50), Pseudomonas aeruginosa (Shen et al., 1981, Infect. Immun.32:967-68), and Mycoplasma pneumoniae (Seggev et al., 1996, Ann. AllergyAsthma Immunol. 77:67-73). IgE antibodies specific for NTHi have alsobeen identified in the serum of patients with CB (Kjaergard et al.,1996, APMIS 104; 61-67; Tee and Pepys, 1982, Clin. Allergy 12:439-50)and cystic fibrosis (Tee and Pepys, 1982, Clin. Allergy 12:439-50).

In a study of patients with bronchial asthma, IgE antibodies to NTHiwere found in 29% of patients. Antibodies to NTHi and/or Streptococcuspneumoniae were also present in 22% of patients with no other IgEmediated hypersensitivity. However, higher levels of IgE bacterialantibodies were found in patients with demonstrable IgE antibodies tovarious inhalant antigens (suggesting an allergic phenotype) (Pauwels etal., 1980, Allergy 157:665-9). While it has been hypothesized thatbacterial infections may play a role in the induction and exacerbationof extrinsic asthma, it has been considered that exacerbation of asthmais predominantly triggered by viral infection. Indeed, the clinicaleffect of bacterial vaccines in the treatment of asthma has beenquestioned, leading to international World Health Organization (WHO)recommendations that bacterial vaccines have no role in modern asthmatreatment.

Despite massive amounts of research focused on therapeutic asthmaintervention and treatment, the condition remains a major, costly andgrowing problem in modern Westernized societies.

Citation or identification of any reference in this application shallnot be construed as an admission that such reference is available asprior art to the present application.

SUMMARY

A novel form of immunotherapy was developed, which involved the oraladministration of inactivated non-typeable Haemophilus influenzae(NTHi), to stimulate the common mucosal system. Protection against acuteexacerbations in COPD was associated with reduction in density of NTHiwithin sputum. These early studies used a poorly characterized NTHiisolate. A combination of selection assays was used to identify one NTHiisolate (which, unless otherwise indicated, is referred tointerchangeably herein as NTHi164, HI-164, isolate 164 or strain 164)that induced broad cross-protection.

Applicants have additionally surprisingly discovered that vaccinationwith HI-164 is useful for treating asthma and reduces the need foranti-asthma therapy.

Accordingly, the present application is directed to vaccines containingHI-164 and similar strains of NTHi, and their uses to treat diseasesinvolving acute episodes of colonization of the mucosal system, such asthe mucosal system of the lower airways. The present application is alsodirected to vaccines containing HI-164 and similar strains of NTHi, andtheir uses to treat pulmonary diseases such as asthma, COPD, and chronicbronchitis that is not associated with COPD. In certain embodiments, thevaccine is a monobacterial vaccine comprising one or more strains NTHiand/or one or more immunogenic fractions of said species of NTHi.

The NTHi strains useful in the vaccines of the present application arecharacterized by a combination of any (e.g., two, three, four, five, sixor all) of the following characteristics:

(1) Lacks a B capsule gene;

(2) Is biotype I;

(3) Lacks a beta-lactamase gene;

(4) Grows aerobically;

(5) Is capable of eliciting a mucosal Haemophilus influenzae-specificIgA response, as reflected by an at least 10%, at least 20%, at least50%, at least 70%, at least 100%, or other statistically significantincrease in Haemophilus influenzae-specific IgA in the saliva;

(6) Is capable of stimulating Haemophilus influenzae-specific T cells inPeyer's patches, as reflected by an at least 10%, at least 20%, at least50%, at least 70%, at least 100%, or other statistically significantincrease in lymphokine production by T-cells from Peyer's patches inresponse to exposure to Haemophilus influenzae);

(7) Encodes an outer membrane protein 2 that displays at least 90%, atleast 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequenceidentity with the outer membrane 2 protein of NTHi-164;

(8) Encodes an outer membrane protein 26 that displays at least 90%, atleast 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequenceidentity with the outer membrane 26 protein of NTHi-164;

(9) Expresses an outer membrane protein 2 that runs as a doublet onsingle dimensional polyacrylamide gel electrophoresis;

(10) Expresses an outer membrane protein 2 that has a pI of 9.1-9.3,most preferably a pI of 9.2;

(11) Expresses prominently on a two-dimensional gel of a whole cellextract at least five, at least 10 or at least 15 of the proteins listedin Table 11A;

(12) Expresses prominently on a two-dimensional gel of a whole cellextract at least two or at least three of the proteins listed in Table11B;

(13) Expresses prominently on a two-dimensional gel of a whole cellextract at least three, at least four, at least five, at least seven orat least ten of the proteins listed in Table 11A, but not listed inTable 11C;

(14) Expresses prominently on a two-dimensional gel of a whole cellextract at least three, at least four, or at least five of the proteinslisted in Table 11A and Table 11D.

As used herein, the term “expresses a protein prominently on atwo-dimensional gel” indicates that the protein is present in a spotsufficient for extraction and MALDI analysis on a two-dimensional gel.

Additionally, the present application provides therapeutic regimens forCOPD patients and asthma patients.

In certain embodiments, the therapeutic regimens for asthma comprise (1)testing a patient, for example a patient who exhibits symptoms ofasthma, for (a) an elevated neutrophil level in saliva, (b) the presenceof a microorganism in sputum or saliva, and/or (c) antibodies specificfor the NTHi and (2) administering a vaccine of the present applicationto a patient who tests positive for one, two or all three parameters. Incertain embodiments, the vaccine administered comprises one, two or moreNTHi strains of the present application and/or an immunogenic fractionof NTHi strains of the present application.

The vaccine of the present application may be a polyvalent vaccine or,more preferably, a monobacterial vaccine.

In certain embodiments, the vaccine of the present application may be inthe form of a tablet, said tablet comprises a core containing saidpopulation or said membrane fraction and an enteric coating surroundingsaid core.

In the vaccine of the present application the weight of the core is 400mg to 500 mg.

In the vaccine of the present application killed bacteria or membranefraction constitutes 7.5% to 15% of the weight of the core of saidtablet.

In the vaccine of the present application killed bacteria or membranefraction constitutes approximately 10% of said core of said tablet.

In the vaccine of the present application said subenteric coatingresults in a 2% to 3% of the weight of the core.

In the vaccine of the present application said enteric coating resultsin a 10% to 12% of the weight of the core.

Any discussion of documents, acts, materials, devices, articles or thelike that has been included in this specification is solely for thepurpose of providing a context for the present application. It is not tobe taken as an admission that any or all of these matters form part ofthe prior art base or were common general knowledge in the fieldrelevant to the present application as it existed anywhere before thepriority date of this application.

The features and advantages of the present application will becomefurther apparent from the following detailed description of embodimentsthereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts growth curves NTHi-164 and a reference strain for thevarious broth media.

FIG. 2 depicts clearance data of bacteria resulting from differentvaccination regimens with NTHi-164 in male Dard Agouti rats.

FIGS. 3A and 3B depict the mean bacteria levels in mice immunized withvarying dose sizes of NTHi-164.

FIG. 4 depicts the mean proliferative response for mesenteric lymph nodelymphocytes from rats immunized with NTHi-164.

FIGS. 5A-5C depicts lymphocyte proliferation in human subjects inresponse to vaccination with HI-164. FIG. 5A demonstrates peripheralblood lymphocyte proliferation to 1 μg HI-164 antigen in vitro. FIG. 5Bshows peripheral blood lymphocyte proliferation to 10 μg HI-164 antigenin vitro. FIG. 5C shows peripheral blood lymphocyte proliferation to PHAin vitro.

FIG. 6 depicts saliva lysozyme (mean±SEM) in human subjects followingvaccination with HI-164.

FIG. 7 shows the effect of vaccination of human subjects with HI-164 onserum IgG levels.

FIG. 8 is a graph showing mean number of NTHi isolated in gargle of aplacebo-dosed human subject study group.

FIG. 9 is a graph showing serum NTHi-specific IgG levels in humansubjects in the placebo group and a treatment group immunized with anoral killed NTHi vaccine.

FIG. 10 is a graph showing saliva NTHi-specific IgG levels in humansubjects in the placebo group and the treatment group immunized with anoral killed NTHi vaccine.

FIG. 11 is a table of total bacterial isolates in sputum followingimmunization with HI-164OV as compared to immunization with placebo.

FIG. 12 is a table of specific bacterial isolates in sputum followingimmunization with HI-164OV as compared to immunization with placebo.

FIG. 13 depicts a comparison of the efficacy of HI-164OV as compared tothe product Broncostat™ (described in PCT Application No. WO86/05691).

FIG. 14 shows the results of single dimension gel electrophoresis ofextracts of HI-164 and HI-166.

FIG. 15 shows the results of single dimension gels of extracts ofvarious Haemophilus influenzae isolates/strains.

FIGS. 16A-16D shows the results of two-dimensional gel electrophoresisof various Haemophilus influenzae isolates/strains. FIG. 16A. HI-164,Plate grown prep 2.

FIG. 16B. HI-166, Plate grown prep 1. FIG. 16C. HI-167, Plate grownprepl. FIG. 16D. HI-164, Outer membrane protein (OMP) preparation.

DETAILED DESCRIPTION

The present application provides NTHi-based vaccines for the treatmentof pulmonary diseases such as asthma and COPD.

The patient needs not have an NTHi infection to benefit from thevaccines of the present application. Thus, in certain aspects, a patientto whom a vaccine of the present application is administered does nothave an NTHi infection or markers indicative of an NTHi infection (e.g.,NTHi-specific antibodies). In other aspects, the patient is positive foran NTHi infection or has markers indicative of an NTHi infection.

Because, in certain embodiments, a vaccine of the present application iscapable of eliciting a non-specific immune response against bacteriaother than NTHi, a patient may be positive for bacteria other than NTHiinfection or has markers indicative of infection by bacteria other thanNTHi. In specific embodiments, the bacteria is one or more of:Staphylococcus aureus; a typeable strain of Haemophilus influenzae;Streptococcus pneumoniae; Escherichia coli; Pseudomonas aeruginosa;Mycoplasma pneumoniae; Haemophilus parainfluenzae; β-HaemolyticStreptococcus spp.; α-Haemolytic Streptococcus spp.; Pseudomonas spp.;Klebsiella pneumoniae; Serratia marcescens; Enterobacter cloacae;Chlamydia pneumoniae; and Moraxella catarrhalis.

The patient to whom the vaccine is administered in accordance with thepresent application will normally be a human being although the vaccinemay also be administered to any suitable mammalian asthma model.

Additional and alternative parameters for a patient to whom the vaccineof the present application is administered are discussed below.

Conventionally in asthma studies, care has been taken to study discretegroups and generally, subjects with clearly defined asthma (e.g.,classical extrinsic asthma) are separated from other groups (e.g., thosewith smoking-related airways disease) leading to the studies beingconducted on defined groups of asthmatics in isolation of other groupsof asthmatics. However, this is an artificial categorization and rather,it is more realistic to view asthma as a spectrum of airways disease asillustrated in Scheme 1.

Various different observations have been made with respect to thesedifferent clinical manifestations of asthma. In brief, these can besummarized as follows:

-   -   The induction of IgE antibody to inhaled antigens (e.g.,        pollens) gives rise to classical allergic asthma in which        allergen-specific IgE binds to mast cells causing degranulation        of the mast cells and releasing of mediators such as histamine        that give rise to allergic symptoms.    -   Colonization of damaged airways and intermittent viral infection        can lead to neutrophil flux into the bronchus (acute bronchitis)        (usually associated with wheeze—thought to follow “inflammation”        of the bronchus).    -   Smoking leading to lung damage can render the subject prone to        infection of the airways.

However, many asthma subjects with clinically diagnosed asthma are‘mixed’ with respect to these components and it is proposed that thisspectrum of asthma disease can be reconciled by recognition thatdifferent pathogenic pathways can lead to asthma and that these pathwayscan co-exist. In particular, without being limited by theory, it isthought by the applicants that the dominant cause of wheeze in manyasthmatics without demonstrable classical allergen hypersensitivity(e.g., negative tests for IgE antibody to house dust, pollens and thelike, and/or whom have elevated eosinophil counts) is due to an IgEantibody mediated reaction to colonizing and/or recurrent exposuremicroorganisms in conjunction with the ability of the microorganism toinduce and activate neutrophils. Specifically, microorganism-basedvaccines can reduce the load of the corresponding microorganism to thesmall airways, and provide effective treatment for so-called “intrinsicasthma”.

In other aspects, and also without being limited by theory, it isthought that some patients who are amenable to treatment by the vaccinesof the present application are allergic to bacteria and have IgEantibodies. It is also believed that in such individuals interactionsmay exist between bacteria and state of allergy to other allergens thatcontribute to the asthmatic state. Thus, in certain aspects, thevaccines of the present application are used to treat a patient who isan allergic asthmatic.

More broadly, benefit from the vaccines of the present application canbe derived by those patients exhibiting one or more parametersindicative of exposure to a microorganism that is capable of colonizingthe airways, such as elevated neutrophil levels (with or withoutelevated eosinophil levels) in saliva, current infection with themicroorganism as for instance indicated by the presence of themicroorganism in sputum or saliva and/or antibodies specific to themicroorganism, and those patients with damaged airways such as arisingfrom smoking (chronic pulmonary obstructive disease (COPD)) or chronicbronchitis (particularly those patients with wheeze). It is recognized,for instance, that patients with damaged airways are highly prone toinfection/colonization by pathogenic microorganisms. While damage toairways classically follows smoking, extrinsic asthma can also damagethe airways (hence, later onset of cough and sputum associated withairways infection). Benefit may also occur in asthmatic patients withcombined mechanisms (e.g., atopic subjects with IgE antibody to amicroorganism that colonizes the airways), and the treatment of asthmaand asthma symptoms in general as a result of decrease or avoidance ofinduction of IgE production resulting from exposure to a vaccinetargeting the microorganism.

Antibody levels can be measured in blood, serum, plasma, sputum orsaliva samples using any suitable conventionally known assay protocolincluding, enzyme linked immunosorbent assay (ELISA) or otherimmunoassay. The antibody tested for can be selected from one or more ofIgA, IgM, IgG and IgE, and subclasses thereof, such as IgG1 and/or IgG3.Total IgE and/or IgE antibody specific to a microorganism that colonizesthe airways will generally be measured in sputum or saliva sample.Neutrophil levels can also be measured in saliva or sputum using anyappropriate conventionally known assay including microscopic evaluationfollowing cell staining. Similarly, any suitable method known in the artcan be employed to determine microorganism counts/level of infection.Antibody levels, neutrophil levels and NTHi counts can be comparedagainst corresponding reference level(s) derived from classicalextrinsic asthmatics (e.g., exhibiting eosinophilic and/orhyper-responsiveness) or for example, a non-asthmatic control or othersuitable reference group. Statistical Methods for differentiating asthmagroups are described in, for instance, Simpson et al., 2006, Respirology11:54-61.

In certain aspects, patients to whom the vaccines of the presentapplication are administered may or may not have COPD and/or symptomsassociated with COPD such as chronic bronchitis and emphysema.

COPD is a slowly progressive disease of the airways that ischaracterized by the gradual loss of lung function. Patients with COPD,which includes chronic bronchitis and emphysema, often require emergencytreatment and sometimes hospitalizations during periods of exacerbationsof their disease. COPD leads to chronic airflow obstruction, which isdefined as a persistent decrease in the rate of airflow through thelungs when the person breathes out (exhales). Symptoms such as wheezingand shortness of breath are relieved when airflow obstruction decreasesby reversing bronchial smooth muscle spasm, inflammation, and increasedsecretions.

Thus, in certain aspects, a patient to whom a vaccine of the presentapplication is administered may have one or more of the followingsymptoms: chronic bronchitis, emphysema, chronic cough, excessive sputumproduction, low blood oxygen levels, severe disabling shortness ofbreath, reduced airflow through the lungs, bronchial smooth musclespasm, bronchial inflammation, and bronchial increased secretions(including mucus plugs).

Severe COPD is generally categorized into two stereotypes—the pinkpuffer and the blue bloater. Most advanced COPD patients have featuresof both stereotypes. The pink puffer is typically an asthmatic,barrel-chested emphysematous patient who exhibits pursed-lip breathingand has no cyanosis or edema. Usually, such a patient uses extrathoracicmuscles to breathe, produces minimal sputum, and experiences littlefluctuation in the day-to-day level of dyspnea. Diaphragmatic excursionsare reduced, and breath and heart sounds are distant. The barrel-shapedchest is non-specific because elderly persons commonly have increasedlung compliance and larger resting lung volumes. The blue bloater istypically overweight, cyanotic, and edematous and has a chronicproductive cough. Elderly blue bloaters are uncommon because bluebloaters often have cor pulmonale, which rapidly leads to death if nottreated appropriately. In certain aspects, a patient to whom thevaccines of the present application are administered exhibit thecharacteristic of a pink puffer and/or a blue bloater.

Chronic hypoxia (reduction of oxygen supply to the body despite adequateblood flow through the body), hypertension, and left ventricularhypertrophy are related conditions which may be symptomatic of COPD orcoincident with COPD. Thus, in certain aspects, a patient to whom avaccine of the present application is administered has chronic hypoxia,hypertension and/or left ventricular hypertrophy.

Cystic fibrosis is an example of an obstructive lung disorder thatresults in bronchiectasis and progressive declines in lung capacity.Thus, in certain aspects, a patient to whom a vaccine of the presentapplication is administered may have cystic fibrosis.

In certain aspects, the patient is positive for a gene or other markerassociated with COPD (see, e.g., US 20080108079 A1 and US 20080044843A1, respectively, each of which is incorporated herein by reference inits entirety). The patient positive for one or more genes or markers mayor may not display symptoms of COPD; i.e., the vaccines of the presentapplications may be administered prior to or following onset of COPDsymptoms.

In certain aspects, the patient to whom the vaccines of the presentapplication are administered does not have asthma. In a specificembodiment, the patient does not have extrinsic asthma. In anotherspecific embodiment, the patient does not have intrinsic asthma.

According to the present application, treatment of asthma encompassesthe treatment of patients already diagnosed as having any form of asthmaat any clinical stage or manifestation; the delay of the onset orevolution or aggravation or deterioration of the symptoms or signs ofasthma; preventing and/or reducing the severity of nighttime and/ordaytime asthma attacks; improving lung capacity; preventing a reductionin lung capacity of asthmatic patients; preventing or limiting adverseexacerbations; preventing or limiting hospital admissions from asthmasymptoms; and/or reducing or limiting the need for antibiotics,steroids, bronchodilators or other medications.

Advantageously, administration of a vaccine in accordance with one ormore embodiments of the present application can lead to a reduction inIgE antibodies and/or a reduction in the symptoms or severity of theasthma (e.g., intrinsic or neutrophilic asthma) in the patient.

According to the present application, treatment of COPD encompasses thetreatment of patients already diagnosed as having any form of COPD atany clinical stage or manifestation; the delay of the onset or evolutionor aggravation or deterioration of the symptoms or signs of COPD;preventing and/or reducing the severity of episodes of wheezing and/orshortness of broth; improving lung capacity; preventing a reduction inlung capacity patients; preventing or limiting adverse exacerbations;preventing or limiting hospital admissions from COPD symptoms; and/orreducing or limiting the need for antibiotics, steroids, bronchodilatorsor other medications.

The vaccine can be administered in accordance with any regimen suitablefor generating an effective immune response against a microorganisminfection. The vaccine of the present application can be administered asa single dose or, where desired or necessary, the initial dose can befollowed by boosters at several days, several weeks, or several monthsor years following the initial dose.

In an exemplary embodiment, a single dose of the vaccine can beadministered once per year pre-winter. Optionally, one or more “booster”doses of the vaccine administered at an interval of a number of weeks ormonths may also be given. Alternatively, a number of doses of thevaccine may be administered over the course of a number of weeks inorder to generate an effective immune response against infection and/orcolonization by a microorganism that is capable of colonizing theairways such as NTHi.

Each dosage administered to a patient can consist of one unit dose (asdescribed below), or more or less. The specific dosage mounts effectivefor therapeutic use will depend on, e.g., the immunogenic component ofthe vaccine (as described below), the weight and general state of healthof the patient, the judgment of the prescribing physician, and theproposed mode of delivery and nature of the vaccine (e.g., capsule,powder, liquid, aerosol delivery, tablets, enterically coated tabletsetc.).

The vaccines of the present application may be administered using anydesired route of administration, including but not limited to, e.g.,subcutaneously, intravenously, intramuscularly or intradermally,although mucosal administration is preferred. Mucosal routes ofadministration include, but are not limited to, oral, rectal and nasaladministration. Preparations for mucosal administrations are suitable invarious formulations as described below. The route of administration canbe varied during a course of treatment.

The vaccine utilized in a method of the present application willtypically contain whole killed or inactivated (e.g., attenuated) NTHiisolate(s) (e.g., formalin-killed) according to the present application.However, soluble or particulate antigen comprising or consisting ofouter cell membrane and/or surface antigens of the microorganism can beutilized as well, or instead of, whole killed NTHi.

Soluble and/or particulate antigen can be prepared by disrupting killedor viable selected NTHi isolate(s). A fraction for use in the vaccinecan then be prepared by centrifugation, filtration and/or otherappropriate techniques known in the art. Any method which achieves therequired level of cellular disruption can be employed includingsonication or dissolution utilizing appropriate surfactants andagitation, and combination of such techniques. When sonication isemployed, the isolate can be subjected to a number of sonication stepsin order to obtain the required degree of cellular disruption orgeneration of soluble and/or particulate matter of a specific size orsize range.

In specific embodiments, the immunogenic components are killed cellsand/or an immunogenic fraction of NTHi species of the presentapplication.

The lack of a beta-lactamase gene is an optional feature of the NTHistrains of the present application. Beta-lactamases are enzymes producedby some bacteria and are responsible for their resistance to beta-lactamantibiotics like penicillins, cephalosporins, cephamycins, ertapenemsand carbapenems. Beta-lactam antibiotics are typically used to treat abroad spectrum of gram positive and gram-negative bacteria. Becausebeta-lactamase expression may result in antibiotic resistance, thepresence of a beta-lactamase gene is generally not preferred during themanufacture and administration of killed pathogens such as NTHi. Thelack of a beta-lactamase gene allows the organism to be controlled withbeta-lactam antibiotics should the need arise during manufacturing or anadverse event in a patient.

The non-typeable H. influenzae isolate HI-164, deposited under theprovisions of the Budapest Treaty with the National MeasurementInstitute in Melbourne, Australia as of Aug. 13, 2008 and assignedaccession no. V08/021002, as is particularly suitable for use invaccines of the present application. Another suitable strain isnon-typeable H. influenzae isolate HI-167, deposited under theprovisions of the Budapest Treaty with the National MeasurementInstitute in Melbourne, Australia as of Aug. 13, 2008 and assignedaccession no. V08/021003.

In one or more embodiments, the outer cellular membrane fraction ormembrane protein(s) of the selected NTHi strain(s) will be utilized asthe immunogenic component of a vaccine of the present application.

Immunogenic proteins and peptides of NTHi have been described. In aspecific embodiment, an NTHi outer membrane protein (“OMP”) fraction orOMP protein is used as an immunogenic component of a vaccine of thepresent application. NTHi OMPs include OMP P6, a highly conserved 16-kDalipoprotein (Nelson et al., 1988, Infect. Immun. 56:128-134) that is atarget of human bactericidal antibody and induces protection both inhumans and in animal models. In chronic pulmonary obstructive disease(COPD), OMP P6 has been shown to evoke a lymphocyte proliferativeresponse that is associated with relative protection from NTHi infection(Abe et al., 2002, Am. J. Respir. Crit. Care Med. 165: 967-71).Accordingly, OMP P6 or any other suitable outer membrane NTHi proteins,polypeptides (e.g., P2, P4 and P26) or antigenic peptides of suchproteins may suitable be used as the immunogenic components of thevaccines of the present application, either in isolated and purifiedform or as a component of a cellular fraction, such as an OMP fraction.

In a certain aspect, the immunogenic protein or peptide is OMP P26 or animmunogenic fragment thereof. In another aspect, the immunogenic proteinor peptide is OMP P2 or an immunogenic fragment thereof. In specificembodiments, the vaccine comprises (1) an OMP P26 protein or immunogenicfragment thereof that has at least 99% or at least 99.5% sequenceidentity with the OMP P26 protein of NTHi-164, and/or (2) an OMP P2protein or immunogenic fragment thereof that has at least 99% or atleast 99.5% sequence identity with the OMP P2 protein of NTHi-164.

A vaccine of the present application will typically comprise the cellsof the selected immunogenic component (i.e., NTHi isolate(s) and/orcellular fractions and/or isolated or purified proteins and/or peptides)in an amount of from about 0.1% to 100% w/w of the vaccine composition,more preferably in an amount of from 1% to 50% of w/w of the vaccinecomposition.

For whole cell killed vaccines, the unit dose will typically be in arange of from about 10⁹ to about 10¹² killed cells, more preferably fromabout 10⁹ to about 10¹¹ killed cells, and most preferably about 10¹⁰ toabout 10¹¹ killed cells.

For vaccines made of cellular fractions of NTHi, the unit dose will befractionated from about 10⁹ to about 10¹⁴ cells, more preferablyfractionated from about 10¹⁰ to about 10¹³ cells, and most preferablyfractionated about 10¹⁰ to about 10¹² cells.

For vaccines containing isolated or purified proteins and peptidefragments, the unit dose is generally 50-75 mg, 75-100 mg, 100-125 mg,125-150 mg, 150-175 mg, 175-200 mg or more.

The optimum dosage of the vaccine can be determined by administeringdifferent dosages to different groups of test mammals, prior tosubsequently infecting the animals in each group with the microorganism,and determining the dosage level required to achieve satisfactoryclearance of the pathogen.

A vaccine of the present application may also comprise one or morepharmaceutically acceptable carriers and/or adjuvants. Exemplaryadjuvants that may be used are further detailed below. Typically,although not exclusively, the preferred oral vaccine formulation isnon-adjuvanted.

Actual methods for preparing the vaccine formulations of the presentapplication will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington'sPharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa.(1980).

The vaccines of the present application are generally provided incompositions with pharmaceutically acceptable carriers. Variouspharmaceutically acceptable carriers are well known in the art. As usedherein, “pharmaceutically acceptable carriers” includes any materialwhich, when combined with an active ingredient of a composition, allowsthe ingredient to retain biological activity and preferably does notcause disruptive reactions with the subject's immune system.

In general, the vaccines can be prepared in various forms, such asgranules, tablets, pills, suppositories, capsules (e.g., adapted fororal delivery), microbeads, microspheres, liposomes, suspensions,salves, lotions and the like. The vaccine itself can be a freeze-driedor lyophilized vaccine reconstituted utilizing a physiologicallyacceptable buffer or fluid. The vaccine can also contain one or moreanti-caking agents, preservatives such as thimerosal or which areotherwise suitable for the proposed mode of administration, stabilizerssuch as amino acids and sugar moieties, sweetening agents such sucrose,lactose or saccharin, surfactants, pH buffering agents and pH modifierssuch sodium hydroxide, hydrochloric acid, monosodium phosphate and/ordisodium phosphate, a pharmaceutically acceptable carrier such asphysiologically saline, solvents and dispersion media and isotonicpreparations.

The vaccine is advantageously presented for oral administration, forexample in a lyophilized encapsulated or tabletted form. It isrecognized that the immunogenic component of a vaccine of the presentapplication, when administered orally, is preferably protected fromdigestion. This can be accomplished either by mixing or packaging theimmunogenic component in an appropriately resistant carrier, such as aliposome, or within an enteric coating. The preparations may also beprovided in controlled release or slow-release forms.

Preferably, the oral formulation is in the form of a capsule or tablet.Such capsules and tablets may be provided with an enteric coatingcomprising, for example, Eudragate “S” (Trade Mark), Eudragate “L”(Trade Mark), cellulose acetate, cellulose phthalate orhydroxypropylmethyl cellulose. Other carriers suitable for formulatingcapsules or tablets include binding agents (e.g., pregelatinized maizestarch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers(e.g., lactose, microcrystalline cellulose or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc or silica);disintegrants (e.g., potato starch or sodium starch glycolate); andwetting agents (e.g., sodium lauryl sulphate). The tablets may be coatedby methods well-known in the art. Preparations for oral administrationmay be suitably formulated to give controlled release of the immunogeniccomponent.

These capsules and tablets may be used as such, or alternatively, thelyophilized material may be reconstituted prior to administration, e.g.,as a suspension.

As an alternative to capsules and tablets, the oral vaccine may be inliquid form, for example, solutions, syrups or suspensions, or may bepresented as a drug product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid).

In order to protect the immunogenic component of the vaccine fromgastric acidity, a sodium bicarbonate preparation may be advantageouslyadministered before each administration of the vaccine.

The vaccine may also be formulated for administration by inhalation orinjection.

Except insofar as any conventional media or agent is incompatible withthe immunogenic component of a vaccine of the present application, orthe proposed mode of administration, their use the vaccines that can beemployed in methods embodied by the present application is specificallyencompassed.

Supplementary active agents for boosting the immune response includingfor instance, probiotic microorganisms, fractions and biologicalproducts thereof, and appropriate cytokines, can also be included to thevaccine.

A vaccine of the present application optionally comprises one or moreadjuvants. Examples of suitable adjuvants are presented herein below.

Suitable adjuvants include mineral salt adjuvants or mineral salt geladjuvants. Such mineral salt and mineral salt gel adjuvants include, butare not limited to, aluminum hydroxide (ALHYDROGEL, REHYDRAGEL),aluminum phosphate gel, aluminum hydroxyphosphate (ADJU-PHOS), andcalcium phosphate. Other suitable adjuvants include immunostimulatoryadjuvant. Such class of adjuvants include, but are not limited to,cytokines (e.g., interleukin-2, interleukin-7, interleukin-12,granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-γ,interleukin-1β (IL-1β), and IL-1 (3 peptide or Sclavo Peptide),cytokine-containing liposomes, triterpenoid glycosides or saponins(e.g., QuilA and QS-21, also sold under the trademark STIMULON,ISCOPREP), Muramyl Dipeptid (MDP) derivatives, such asN-acetyl-muramyl-L-threonyl-D-isoglutamine (Threonyl-MDP, sold under thetrademark TERMURTIDE), GMDP,N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine,N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine,muramyl tripeptide phosphatidylethanolamine (MTP-PE), unmethylated CpGdinucleotides and oligonucleotides, such as bacterial DNA and fragmentsthereof, LPS, monophosphoryl Lipid A (3D-MLAsold under the trademarkMPL), and polyphosphazenes. Yet other suitable adjuvants includeparticulate adjuvants, including, but not limited to, emulsions, e.g.,Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, squalene orsqualane oil-in-water aduvant formulations, such as SAF and MF59, e.g.,prepared with block-copolymers, such as L-121(polyoxypropylene/polyoxyethylene) sold under the trademark PLURONICL-121, Liposomes, Virosomes, cochleates, and immune stimulating complex,which is sold under the trademark ISCOM. Yet other suitable adjuvantsare microparticulate adjuvants such as, but not limited to,biodegradable and biocompatible polyesters, homo- and copolymers oflactic acid (PLA) and glycolic acid (PGA), poly (lactide-co-glycolides)(PLGA) microparticles, polymers that self-associate into particulates(poloxamer particles), soluble polymers (polyphosphazenes), andvirus-like particles (VLPs) such as recombinant protein particulates,e.g., hepatitis B surface antigen (HbsAg).

A preferred class of adjuvants are mucosal adjuvants, including but notlimited to heat-labile enterotoxin from Escherichia coli (LT), choleraholotoxin (CT) and cholera Toxin B Subunit (CTB) from Vibrio cholerae,mutant toxins (e.g., LTK63 and LTR72), microparticles, and polymerizedliposomes.

In certain aspects, the adjuvant is an adjuvant that activates a Th1immune response. Preferably, the adjuvant does not activate a Th2 immuneresponse, although adjuvants that activate a Th2 immune response arewithin the scope of the present application.

The vaccines of the present application can be formulated in anysuitable manner. In general, the vaccines of the present application canbe administered orally, nasally, nasopharyngeally, parenterally,enterically, gastrically, topically, transdermally, subcutaneously,intramuscularly, in tablet, solid, powdered, liquid, aerosol form,locally or systemically, with or without added carriers.

A vaccine of the present application can be administered as a capsule ortablet, as a dry powder or in liquid form. Administration can forexample be achieved by injection (cg, subcutaneous, or intravenous),orally such as by dosage unit form (e.g., tablet, capsule or dosedliquid form), or by inhalation.

In certain aspects, a vaccine of the present application is administeredin a manner that allows the immunogenic component to reach a lymphoidtissue, more preferably a secondary lymphoid tissue, and most preferablya mucosa-associated lymphoid tissue. In certain embodiments, themucosa-associated lymphoid tissue is BALT (bronchus-associated lymphoidtissue), NALT (nose-associated lymphoid tissue), LALT (larynx-associatedlymphoid tissue), or GALT (gut-associated lymphoid tissue). Mostpreferably, administered by way of an enterically coated tablet to allowdelivery of the immunogenic component of the vaccine to Peyer's patches,which are aggregations of lymphoid tissue that are predominantly foundin the lowest portion of the ileum, and other gut-associated lymphoidtissue (GALT) in the patient's gut.

Described below are combinatorial methods in which the vaccines of thepresent application can be utilized. The combinatorial methods of thepresent application involve the administration of at least two agents toa patient, the first of which is a vaccine targeting a microorganismaccording to the present application, and the second of which is asecond therapeutic agent.

The combinatorial therapy methods of the present application can resultin a greater than additive effect, providing therapeutic benefits whereneither the vaccine nor second therapeutic agent administered in anamount that is alone effective for treatment of asthma.

In the present methods, the vaccine and the second therapeutic agent canbe administered concurrently or successively. As used herein, thevaccine and the second therapeutic agent are said to be administeredconcurrently if they are administered to the patient on the same day,for example, simultaneously, or 1, 2, 3, 4, 5, 6, 7 or 8 hours apart. Incontrast, the vaccine and the second therapeutic agent are said to beadministered successively if they are administered to the patient on thedifferent days, for example, the vaccine and the second therapeuticagent can be administered at a 1-day, 2-day or 3-day, one-week, 2-weekor monthly intervals. In the methods of the present application,administration of the vaccine can precede or follow administration ofthe second therapeutic agent.

As a non-limiting example, the vaccine and second therapeutic agent canbe administered concurrently for a period of time, followed by a secondperiod of time in which the administration of the vaccine and the secondtherapeutic agent is alternated.

Because of the potentially synergistic effects of administering avaccine and a second therapeutic agent, such agents can be administeredin amounts that, if one or both of the agents is administered alone,is/are not effective for treating asthma or COPD.

Most people with persistent asthma use a combination of long-termcontrol medications and quick-relief medications, taken with a hand-heldinhaler. Asthma symptoms triggered by airborne allergens, such as pollenor pet dander, are also treated with allergy medications.

Accordingly, suitable second therapeutic agents include long-termcontrol medications, quick-relief medications, and allergy medications.

Examples of long-term control medications include, but are not limitedto, (1) inhaled corticosteroids such as fluticasone (e.g., FloventDiskus™), budesonide (e.g., Pulmicort™), triamcinolone (e.g.,Azmacort™), flunisolide (e.g., Aerobid™), and beclomethasone (e.g.,Qvar™); (2) long-acting beta-2 agonists (LABAs) such as salmeterol(e.g., Serevent Diskus™) and formoterol (e.g., Foradil Aerolizer™,Oxis™, Performist™ and Brovana′); (3) long acting muscarinic antagonistssuch as tiotropium (e.g., Spiriva™) and ipratropium (e.g., Atrovent™);(4) leukotriene modifiers such as montelukast (e.g., Singulair™)zafirlukast (e.g., Accolate™) and zileuton (e.g., Zyflo CR™); (5) mastcell inhibitors such as cromolyn (e.g., Intal™) and nedocromil (e.g.,Tilade™); (7) theophylline

Examples of quick-relief medications include (1) short-acting beta-2agonists (SABAs) such as albuterol or albuterol sulfate (e.g., as soldunder brand name Xopenex™ and Ventolin™), (2) short acting muscarinicantagonists, and (3) oral and intravenous corticosteroids such asprednisone, methylprednisolone, mometasone furoate (e.g., as sold underbrand name Asmanex™) and ciclesonide (e.g., Aerobid™/Alvesco™).

Examples of allergy medications include (1) immunotherapy, (2)anti-histamines (e.g., Claritin™ and Zyrtec™) and (3) anti-IgEmonoclonal antibodies, such as omalizumab (Xolair™). Examples ofmucolytics include, but are not limited to, Bronchitol™ (a mannitolinhaler) and Mucomyst™ (an acetylcysteine inhaler).

In certain aspects, the second therapeutic agent itself is a combinationproduct, i.e., a product containing more than one active ingredient.Examples of suitable combination products include Symbicort™ (acombination of formoterol and budesonide); Combivent™ (a combination ofatrovent and albuterol); and Advair™ or Seretide™ (a combination ofsalmeterol and fluticasone); a combination of long actingbeta-adrenoceptor agonist such as indacaterol with mometasone; and acombination of long acting beta-2 agonist such as formoterol withmometasone.

BRONCHODILATORS—Medications that help open the airways are a mainstay oftreatment for COPD. Bronchodilators are most commonly given in aninhaled form using a metered dose inhaler (MDI), dry powder inhaler(DPI), or nebulizer. It is important to use the inhaler properly todeliver the correct dose of medication to the lungs, rather than themouth. Any of the bronchodilators described above may be used incombination with the NTHi vaccines of the present application for thetreatment of COPD.

VACCINES—In certain aspects, the second therapeutic agent is a vaccine,such as a pneumococcal vaccine, which helps prevent a certain type ofpneumonia, or an influenza vaccine, which is generally administeredbefore the influenza season, generally in the late fall or early winter.

ANTIVIRAL AGENTS: For COPD patients who get influenza, antiviralmedications may be prescribed. Antiviral medication may also berecommended for people with COPD who have not had an influenza vaccineand are at risk for getting influenza. Thus, in certain aspects, thesecond therapeutic agent is an antiviral agent.

ANTIBIOTICS—Antibiotics are of some benefit in people with a bacterialrespiratory infection who have worsening COPD symptoms. A physician mayorder a sputum analysis (from sputum in the lungs) to determine ifantibiotics are needed in a particular situation. Thus, in certainaspects, the second therapeutic agent is an antibiotic.

OXYGEN—Patients with advanced COPD can have low oxygen levels in theblood. This condition, known as hypoxemia, can occur, even if the persondoes not feel short of breath or have other symptoms. The oxygen levelcan be measured with a device placed on the finger (pulse oximeter) orwith a blood test (arterial blood gas). Patients with hypoxemia may beplaced on oxygen therapy, which can improve survival and quality oflife, depending upon the degree to which the oxygen level in the bloodwas decreased. Thus, in certain aspects, the second therapeutic agent isoxygen.

AUGMENTATION THERAPY FOR SEVERE ALPHA-1-ANTITRYPSIN DEFICIENCY—Peoplewith alpha-1-antitrypsin deficiency as a contributor to their COPD canbe treated with purified alpha-1-antitrypsin that is prepared fromdonated, pooled blood. Thus, in certain aspects, the second therapeuticagent is alph-1-antitrypsin.

OTHER THERAPIES—Certain patients with COPD may be given othertreatments, including: noninvasive ventilatory support (the use of aspecial mask and breathing machine to improve symptoms), anti-anxiety oranti-depressant medications, or morphine-like medications to reduceshortness of breath. Such therapies can constitute the secondtherapeutic agent of the present application.

Example 1: SELECTION OF Non-Typeable H. influenzae STRAIN HI-164

1.1 Background

A number of NTHi isolates, including strain IDs 164, 165, 167 and 168,were collected from 20 patients attending a respiratory clinic at theJohn Hunter Hospital in Newcastle, Australia. The isolates and anotherresearch isolate (strain NTHi-166) were characterized for growthconditions, biotype, serotype, the presence of a B capsule gene and thepresence of the beta lactamase gene. The results are given in Table 1below:

TABLE 1 Growth characteristics of different NTHi isolates Growth Bcapsule Beta Strain ID Condition Biotype Serotype gene lactamaseNTHi-164 Aerobic I Non- Negative Negative typeable NTHi-165 Aerobic INon- Negative Positive typeable NTHi-166 Aerobic I Non- PositiveNegative typeable NTHi-167 Aerobic I Non- Negative Negative typeableNTHi-168 Anaerobic I Non- Negative Positive typeable

The isolates were tested for usefulness as the active component of anoral vaccine in a rodent model of acute respiratory infection. Theisolates were also tested for ability to stimulate human bloodlymphocytes in vitro to secrete IFNγ. On the basis of these testsisolate NTHi-164 has been selected as the NTHi strain for use in theHunter Immunology Ltd HI-164OV oral vaccine. This isolate has propertiesthat make it an ideal choice for use in an oral vaccine to provideprotection against H. influenzae infection.

In both the Examples and the remainder of the specification, the term“HI-164OV” refers to a vaccine tablet comprising HI-164, i.e.formalin-killed lyophilized NTHi-164 active substance.

1.2 Summary of Properties of NTHi-164

1.2.1 Microbiological Identification

H. influenzae identification: Requires X and V factors for growth.Non-typeable identification (i.e., no capsule of any type) and noexpression of B capsule and no gene for B capsule, no β-lactamase.

1.2.2 Growth Characteristics

Grows well in Oxoid™ Tryptone Soya Broth (TSB) supplemented withHaemophilus Test (HTM) supplement. Also grows well in Oxoid™ veggitonebroth with HTM supplement.

The ability of various media to grow H. influenzae NTHi-164 andATCC19418 was examined and results compared. H. influenzae NTHi-164 andH. influenzae ATCC19418 were incubated in 250 mL conical flaskscontaining 100 mL of either

-   -   Tryptone Soya Broth (Code CM129. Lot B.:301377)    -   Tryptone Soya Broth (Code CM129. Lot B.:301377) plus Yeast        Extract (Oxoid Code L21, Lot Ch-B.:856108)    -   Veggitone Vegetable Peptone (Oxoid Code VG0100, Lot 335918)    -   Veggitone Vegetable Peptone (Oxoid Code VG0100, Lot 335918) plus        Yeast Extract (Oxoid Code L21, Lot Ch-B.:856108)

Each medium was supplemented with Haemophilus Test Supplement (HTM),Oxoid Code SR158E. Lot B.:312141. Growth was at 37° C. in ambient airwith approximately 2 rotations of the flasks per second. The stationaryphase was reached after approximately 11 hours of incubation.

All media used supported the growth of both strains of H. influenzae,with the more nutritious TSB plus yeast extract and HTM supplement beingthe preferred formulation. Animal based media (TSB) and vegetable basedmedia (Vegetable Peptone) were approximately equivalent in growthCharacteristics for the 2 isolates.

With the preferred media formulation (TSB with Yeast Extract and HTMsupplement) optical densities for H. influenzae strains NTHi-164 andATCC 19418 plots over time were roughly equivalent, with OD's at 595 nmand 11 hours of incubation for H. influenzae NTHi-164 and ATCC 19418being 0.590 and 0.525 respectively. Please note that other culture mediamay produce similar or better growth results. Growth curves of NTHi-164and ATCC 19418 are shown in FIG. 1.

For large scale production, NTHi-164 can be grown in a medium containing30+/−0.3 g/L tryptone soya broth; 5+/−0.05 g/L yeast extract; and0.20+/−0.01 g/L antifoam and supplemented with nicotinamide adeninedinucleotide (NAD) at 15 mg/ml and haematin at 15/mg/ml. The supplementsare added in volumes of up to 4 mL per L.

1.3 Capacity to Induce Protection in Rodent Models of Infection

These data demonstrate that killed NTHi-164 has the capacity tostimulate a protective immune response. Applicants believe that thiscapacity is related to the pattern of expression of bacterial antigensand/or existence of natural adjuvants, and/or to the capacity for uptakeinto Peyer's patches (which is likely related to the particle size andalso possibly to adhesion proteins).

1.4 HI-164 Protects Against Acute Respiratory Infection in Rats

Rats were immunized on day 0 by a single dose of 3×10⁷, 3×10⁸, or 3×10⁹formalin-killed NTHi-164 (referred to herein as HI-164) delivereddirectly to the intestinal lumen, or followed by one oral dose on day 14or two oral doses on days 14 and 21 (i.e., rats received one, two orthree doses). On day 28 a boost dose (with capacity to boost theintestinal dose but without the capacity to provide protective immunityon its own) was delivered to the lungs by instillation into the trachea.Control rats for each immunisation regimen were immunized with placebo(PBS). On day 35 the rats were infected by intra-tracheal instillationof 5×10⁸ live NTHi-164. Four hours later the rats were killed forsampling. Lungs were lavaged to provide broncho-alveolar lavage (BAL)fluid and the lung was homogenized in PBS to provide a lung homogenate(LH). Serial dilutions of the BAL and LH were prepared and samples ofeach dilution plated out on chocolate agar plates. The plates werecultured overnight at 37° C. in an atmosphere of 5% CO₂. The colonies onthe plate were counted and the number of NTHi-164 (colony forming units,or CFU) in the BAL and LH calculated. The total NTHi-164 CFU in the lungwas determined. The mean CFU in the lungs for the vaccine immunized andplacebo immunized animals was calculated and the clearance of bacteriaprovided by each vaccination regimen was calculated compared to therelevant control group. The results are shown in FIG. 2. Good bacterialclearance was obtained in animals that received one or more doses whenthe dose was 3×10⁸, or 3×10⁹. Good bacterial clearance was obtained inanimals that received two or three doses when the dose was 3×10⁷.

1.5 NTHi-164 Protects Against H. influenzae Respiratory Infection inMice

These experiments demonstrate the capacity of NTHi-164 to protect miceas well as rats. It further demonstrates that the vaccine is mostprotective in a mouse strain that is most susceptible to infection.

C57BL/6 or Balb/c mice were immunized by gavage with PBS or killedNTHi-164 in PBS (dose sizes 5×10⁶, 5×10⁷, and 5×10⁸) on days 0 and 14.Mice were boosted intra-tracheally with a non-immunizing dose (5×10⁵) ofkilled NTHi-164 on day 20 or 21. Mice were infected intra-tracheallywith 1×10⁷ live NTHi-164 on day 27 or 28 and killed 4 h later forsampling. Levels of live Hi-164 in the lungs was measured by platingserial dilutions of bronchoalveolar lavage (BAL) fluid and lunghomogenate (LH) on chocolate agar plates and culturing overnight at 37°C. in 5% CO₂. The colonies were counted, the number of NTHi-164 in theoriginal BAL and LH samples calculated and the total bacteria levels ineach mouse lung calculated. The mean bacteria level in each mouse group(±SEM) is shown in FIGS. 3A and 3B. Unimmunized C57BL/6 mice had higherinfection levels than unimmunized Balb/c mice indicating that Balb/cmice had better innate protection. However the killed NTHi-164 vaccinewas highly effective in C57BL/6 mice suggesting that the specificprotective mechanism was of the Th1-type as C57BL/6 mice have been shownto have a Th1 phenotype (i.e., respond more readily with a Th1-typeimmune response) and the Balb/c mice more of a Th2-phenotype (i.e.,respond more readily with a Th2-type response).

1.6 Capacity to Cross-Protect Against Other H. influenzae Isolates

This example demonstrates the broad cross-protection provided byimmunization with formalin-killed NTHI-164. This would not be predictedby Sethi and Murphy (Sethy & Murphy, 2002, Clinical Microbiology Reviews14(2):336-363) who see the lack of conservation by the major OMP P2 asbeing the cause of apparent lack of cross-protection in man.

Rats were immunized by the intra-tracheal route on days 0 and 14 with avaccine comprising 5×10⁸ killed NTHi-164 in 50 μL PBS. The rats weresubsequently infected on day 21 with one of a variety of H. influenzaestrains that were non-typeable strains of different biotype, or type bstrains. Table 2 shows that protection was provided against all biotypestested of the non-typeable strains. Table 3 shows that protection wasalso provided against 2 of 3 type b strains tested.

TABLE 2 % clearance of NTHi % clearance of NTHi: (CFU in placebo—CFU inactive) × 100/CFU in placebo Infection strain (non-typeable H.influenzae isolates) Hi 289 Biotype Isolate Isolate Isolate Isolate ATCCATCC ATCC ATCC I 1 2 3 4 43041 43095 35092 51997 Vaccine NTHi- NTHi-NTHi- NTHi- NTHi- Biotype Biotype Biotype Biotype strain 166 165 164 168167 VII III VIII V Hi-164 87 82 87 91 83 74 85 78 89

TABLE 3 % clearance of H. influenzae type B % clearance of NTHi: (CFU inplacebo − CFU in active) × 100/CFU in placebo Infection strain (H.influenzae type b isolate) Vaccine strain Hib 8719 ATCC 10211 ATCC 9795NTHi-164 62% 0% 39%

1.7 Induction of Specific Immune Responses in Rodents

This example demonstrates that vaccination with killed NTHi-164 leads tothe induction of NTHi-specific T cells, which are important inprotection of the lung against bacterial infections.

Groups of 5 rats were immunized by instillation into the intestinallumen (exposed by laparotomy) on day 0, of 0.5 mL of

-   -   PBS    -   NTHi 164 specimen at a dose of 5×10⁸ in 5004 PBS    -   NTHi 164 specimen at a dose of 5×10⁹ in 5004 PBS

Rats were sacrificed on day 14 and mesenteric lymph nodes were collectedfor preparation of lymphocyte suspension and in vitro culture (intriplicate) with NTHi 164 antigen at 0.1, 1 and 10 ug/mL for 5 days.During the last 6 h of culture wells were pulsed with ³H-thymidine todetect lymphocytes proliferating in response to antigen stimulation, andcultures were then harvested on filter mats. Mats were placed in bagswith scintillant and radioactivity (counts per minute) for each culturewell, measured in a ß-counter. FIG. 4 shows the mean proliferativeresponse for lymphocytes from each rat group. This demonstrates thatNTHi-164-specific lymphocytes can be detected in the mesenteric lymphnodes following immunization with killed NTHi-164.

1.8 Lack of Toxicity of Killed Isolate in Rodents

These studies provide toxicity data for HI-164OV, which suggest that thevaccine will be safe in man.

Groups of 12 Sprague-Dawley rats were dosed with PBS (placebo), a rattherapeutic dose of 2.25 mg (corresponding to 5×10⁹ killed cells), or ahuman-size dose of 45 mg (corresponding to 1×10¹¹ killed cells) ofHI-164 active substance suspended in PBS (manufacturing batch no.VRI0401). Dosing was on days 0 and 14 and rats were euthanased(pentobarbitone overdose) on day 21 for post-mortem examination andsample collection. The following parameters were measured:

-   -   Clinical assessment daily    -   Body weight weekly    -   Post-mortem examination (perform by veterinarian)    -   Blood biochemistry    -   Blood haematology    -   Histopathology on heart, lung, liver, spleen, kidney, stomach,        pancreas, duodenum, jejunum, ileum, colon, mesenteric lymph        nodes    -   Biochemistry, Haematology and histopathology was performed by        Idexx

Laboratories, Australia)

No differences were found between active treatment groups and theplacebo group for any of these parameters.

1.9 Capacity to stimulate NTHi-specific T cells in human blood

This study suggests that Haemophilus influenzae-specific T cells inhuman blood recognise NTHI-164 and can secrete cytokines such as IFN-γin response. IFN-γ is also involved in the protective mechanism wherebyIFN-γ activates phagocytic cells (macrophages and polymorphonuclearcells) to more actively phagocytose and kill bacteria.

An initial study examined 4 volunteers. On day 1 blood was collectedinto lithium heparin tubes by venupuncture. Peripheral blood mononuclearcells (PBMC) were separated by centrifugation on a Ficoll-Paque densitygradient. PBMC were cultured in serum-free medium in wells of aflat-bottom 96 well plate. Cultures were unstimulated (medium only) orstimulated with 2×10⁶ and 2×10⁸/mL of killed HI-164 or 10 μg/mL, 1 μg/mLand 0.1 μg/mL of NTHi 164 sonicate antigen for 3 days in an incubatorset at 37° C. plus 5% CO₂. On day 3 culture supernatants were collectedfrom each well into 1.5 mL eppendorf tubes, clarified by centrifugationfor 2 min at 12000 rpm (g), aliquoted and stored at −70° C. until levelsof IFN-γ, IL-10 and IL-12 were measured by ELISA.

Levels of IFN-γ obtained from NTHi 164 stimulated PBL culturesupernatants are shown in Table 4 below:

TABLE 4 Levels of IFN-γ (pg/mL) in the culture supernatant of PBLstimulated with NTHi 164 antigen and PHA Antigen MD PH PC RC Mean SD  10ug/ml (sonicated NTHi) 297 58 150 101 152 103   1 ug/ml (sonicated NTHi)54 35 55 35 45 11 0.1 ug/ml (sonicated NTHi) <15 <15 <15 20 20 PHA(5ug/ml) <15 30837 9568 8617 16341 12563 2 × 10⁸(killed NTHi) 1738 11934533 514 1994 1764 2 × 10⁶(killed NTHi) 207 257 491 72 257 174 cellsonly <15 <15 <15 <15 AIM-V only <15 <15 <15 <15

A second study examined seven subjects recruited for a safety andimmunogenicity study of an HI-164 vaccine. On day 0, prior to takingstudy medication, 10 mL of heparinized blood was received from visit 1of Clinical Trial HHI003 subjects 001 ESY, 002 SP, 003 MBW, 004 JMW, 005JDL, 006 RGB and 007 MA. Peripheral blood lymphocytes (PBL) wereisolated on a Ficoll-Paque density gradient, washed, and resuspended inAIM-V culture medium with added amphotericin B (0.25 ug/mL), 1M HepesBuffer and 2-mercaptoethanol (5×10⁻⁵M)(SOP: IMM027).

Cells were cultured for 5 days in flat bottom 24 well plates at 1×10⁶cells/well in 1 mL of medium. Cells were stimulated with twoconcentrations of NTHi 164 antigen (10 μg/mL, 1 μg/mL) and PHA (5 μg/mL)(SOP: IMM011). PHA is a pan T cell stimulator.

On day 5, the culture supernatants were collected from each well,clarified by centrifugation then aliquoted and stored at −70° C. untilIFN-γ levels in culture supernatant were measured by ELISA. The resultsare shown in Table 5 below.

TABLE 5 Levels of IFN-γ (pg/mL) in the culture supernatant of PBLstimulated with NTHi 164 antigen and PHA Culture 001 002 003 004 005 006007 stimulant ESY SP MBW JMW JDL RGB MA 10 μg/mL NTHi 368 128 101 169356 565 334 164  1 μg/mL NTHi 130 287 24 111 156 86 35 164  0 μg/mL NTHi0 0 0 61.3 0 0 0 164  5 μg/mL PHA 0 9 0 38 9457 626 6 AIM V 0 0 0 0 0 00

NTHi 164 (10 μg/mL and 1 μg/mL) stimulated production of IFN-γ in PBLculture supernatant of all subjects.

Capacity to Induce Specific T Lymphocyte Immune Response in Humans

Subjects recruited to a placebo-controlled safety and immunogenicitystudy were examined for specific immune response induction. FIG. 4 showsthe induction of specific T lymphocytes that occurred in the vaccine andplacebo treatment groups over the course of the study. The specific Tlymphocytes in the blood of the placebo patient group increased over theperiod of the study (FIGS. 5A, 5B). This response was most likelyinduced by exposure to H. influenzae bacteria during this period. Inpatients given the active tablet treatment the induction of specificlymphocytes was enhanced suggesting a boosting of the naturally-inducedimmune response. An enhanced response to PHA in vitro was also observedin blood lymphocytes from the HI-164OV group (FIG. 5C).

1.10 HI-164OV Treatment Reduces Salivary Lysozyme

The observation of reduction in salivary lysozyme in the HI-164OVtreated group suggests a reduction in inflammation. (FIG. 6)

1.11 Capacity of a Killed NTHi-164 Oral Vaccine (HI-164OV) to PreventBacteria Reaching the Lower Airways

The same subjects as described above were examined for relationshipbetween serum IgG and number of visits at which NTHi was detected inthroat gargles. The mean (±SEM) log change in serum NTHi-specific IgGbetween visits 1 and 6 was plotted against the number of visits(classified as either 0-1 visits or 2-4 visits) at which H. influenzaewas detected in the gargle (FIG. 7A). In the placebo group high levelsof serum NTHi-specific IgG were associated with live NTHi detection at 2or more visits. No such association was apparent with subjects treatedwith the active HI-164OV tablets. A plot of median change in serum IgGfrom base is shown in FIG. 7B for all visits 2-7. Again thisdemonstrates an increase in serum NTHI-specific IgG in the placebotreatment group and a reduction in serum NTHi-specific IgG in theHI-164OV treatment group. This data was interpreted as the activetreatment preventing bacteria in the upper airways from entering thelower airways and inducing H. influenzae-specific IgG in the serum.Median change from baseline (visit 1) in serum NTHi-specific IgG forsubjects with 0-1 or 2-6 visits at which H. influenzae was detected inthe gargle is shown in FIG. 7B.

1.12 Capacity of a Killed NTHi-164 Oral Vaccine (HI-164OV) to ProvideProtection Against Severity of Exacerbations in Patients withMild-to-Moderate or Moderate-to-Severe Airway Disease.

In a double-blind, placebo-controlled parallel clinical study examiningthe efficacy of the HI-164OV vaccine 164 patients with mild-to-moderateor moderate-to-severe airway disease were dosed with 18 tabletscontaining 45 mg (equivalent to 10¹¹ killed bacteria). Two tablets perday were given on days 1, 2, 3, 29, 30, 31, 57, 58, 59. General health,adverse events, acute exacerbations of chronic bronchitis and severityof these exacerbations was monitored. It was found that the activetreatment group had a considerable reduction in hospitalisations(largely due to exacerbations of bronchitis) compared to the placebocontrol group (7 patients with 10 hopitalisations in the placebo groupand 1 patient with 1 hospitalisation in the HI-164OV group). These dataare indicative of the protective capacity of this vaccine activesubstance when used as an oral vaccine.

Example 2: CLINICAL BENEFITS OF HI-164oV

A placebo-controlled double-blind clinical study was performed in which64 subjects on the basis of having smoked at least 10 cigarettes per dayfor the past two years were recruited and allocated to oral NTHi therapyor placebo treatment groups in a double-blind study. Subjects wererandomized into placebo and active groups and were given three coursesof study medication at monthly intervals. Each course consisted of twotablets per day for three days. The active tablets each contained 45 mgof formalin-killed NTHi (equivalent to 10¹¹ killed bacteria per activetablet). Blood, saliva, gargles, throat swabs, and nasal swabs (formicrobiological assessment) were collected at seven fortnightly visits.

1.13 Detection of NTHi and Measurement of NTHi-Specific IgG

Surprisingly, measurements in the placebo-treated and vaccine-treatedgroups over the winter period detected NTHi in both groups indicatingrandom exposure to the bacterium. FIG. 8 shows the mean level of NTHi inthe gargles of the placebo group at each visit.

NTHi-specific IgG was measured in serum and saliva by ELISA assay.Briefly, wells of 96-well Nunc Maxisorp plates were coated with H.influenzae 164 sonicate antigen preparation. After incubation overnightat 2-8° C. the plates were washed and samples of serum or saliva atvarious dilutions were added. Following incubation at room temperaturefor 60 minutes, the plates were washed and horse-radishperoxidise-conjugated anti-human IgG antibody (Chemicon catalogue numberAP112P) was added. After incubation for a further 60 minutes at roomtemperature the plates were washed and TMB substrate (Biomediq cataloguenumber 50-76.00) was added prior to an additional incubation for 10minutes at room temperature and the reaction being stopped by additionof 1M phosphoric acid. Absorbance was read on a BioRad microplate readeron dual wavelength mode with a primary filter of 450 nm and referencefilter of 655 nm. A standard curve was used to determine the ELISA unitsin each sample.

Levels of NTHi-specific IgG in serum and saliva in the placebo groupwere higher and more variable than the levels in the vaccine-treatedgroup (see FIG. 9 and FIG. 10). Applicants believe this because NTHireaching the lower airways in the placebo group results in systemicproduction of IgG and that was essentially prevented from reaching thelower airways in the vaccine treated group. To test this, plots wereprepared of relationship between the number of visits at which NTHi wasdetected in the gargle and the median change in serum IgG between visits1 and 6 (FIG. 7). Placebo and active subjects were grouped according towhether they had 0-1 visits or 2-6 visits where NTHi was found in thegargle. In the placebo group, positive increases in serum IgG wereassociated with increased number of NTHi detections. This was not foundin the active treatment group. The difference between the placebo andactive change in IgG was statistically significant (p=0.0186) indicatingthe serum IgG in the placebo group was indeed generated by NTHi as aresult of the bacteria reaching the lower airways. Moreover, the moreNTHi present in the placebo washings, the higher the IgG antibody level.This is also believed to apply to the appearance of salivary NTHispecific IgG in the placebo group.

1.14 Discussion

Serum IgG antibody as a marker for the efficacy of the vaccine wasmeasured. An apparent lack of an IgG response in the vaccine-treatedgroup was found while the placebo treated group of patients showed anincrease in serum IgG. Without being limited by theory, it is believedby the applicants that the increase in IgG observed in the placebo groupis reflecting an immune response to infecting bacteria reaching thelower airways where uptake of the bacteria by antigen-presenting cellsand transport to draining lymph nodes induces an anti-bacterial IgGresponse. In contrast, the lack of such a response in thevaccine-treated group indicates that the bacteria are being essentiallyprevented (by a mucosal vaccine-specific immune response) from reachingthe lower airways. A comparison of the IgG response in subjects withNTHi detected in the upper airways at low frequency (detected at 0-1visits) or at high frequency (detected at 2-6 visits) also showed theincrease in IgG in the placebo group but not in the active (vaccine)treatment group. This suggests that serum IgG measurement following oralvaccination with NTHi reflects exposure to infection and the degree towhich this is prevented by mucosal immunization. The saliva IgG responsereflected that seen in the serum.

Overall, this study demonstrates detection of NTHi in the upperrespiratory tract of subjects in both the treatment and placebo groups,and that treatment with oral killed NTHi vaccine therapy led to areduction NTHi-specific IgG in serum and saliva in the treatment groupindicating the vaccine was successful in limiting or preventing accessof NTHi to the lower airways (i.e., less allergen to initiate asthma oraggravate COPD).

Thus, only in the placebo group did NTHi access the lower airways asevidenced by stimulation of IgG antibody, and oral ‘immunization’ withNTHi vaccine reduced NTHi allergen in the airways.

Example 3: Killed NTHi-164 Vaccine (HI-164OV) Orally Administered toSubjects with Mild, Moderate or Severe Airway Disease Reduced Usages ofAnti-Asthma Therapy

One hundred and forty human subjects with mild-to-moderate ormoderate-to-severe airway disease were recruited into a double blind,placebo-controlled study to assess the effect of an oral killednon-typeable Haemophilus influenzae (NTHi) vaccine on number andseverity of wheezy reversible airways obstruction, and usage ofconcomitant medication as well as the presence of NTHi and otherbacteria in the airways.

A reduction in use of anti-asthma-type medication (bronchodilators,steroids etc) and reduced infection by NTHi was found in the treatmentgroup compared to the control group. In particular, a specific reductionof NTHi within the airways of subjects with high IgE antibody levels(serum and secretions) to NTHi, and a reduction in asthma symptoms witha consequential reduction in the need for asthma medication wasobtained.

1.15 Subjects and Clinical Trial

This study was a multi-center, double blind, placebo controlled trialconducted at four sites under the supervision of a Clinical ResearchOrganisation. These sites were: Hollywood Private Hospital, Perth, Wash.(Dr M. K. Tandon); Sir Charles Gairdner Hospital, Perth, Wash. (Dr M.Phillips); Royal Perth Hospital, Perth, Wash. (Assoc. Prof. G. Waterer)and the University of Newcastle, Newcastle, NSW (Prof R. Clancy).Identical protocols were accepted by the ethics committees of eachinstitution, and the studies conformed to the guidelines of thesecommittees. The main admission criterion was a history of two or moreepisodes per year, for two consecutive years (an ‘episode’ was definedas ‘an increase in cough and purulent sputum from background levels’).Subjects were stratified into two groups: i) patients with an FEV₁ of≤50% of predicted normal value (38 subjects), and ii) patients with FEV₁of >50% of predicted normal value (102 subjects). This second groupincluded patients with COPD, patients with a primary diagnosis ofbronchiectasis and patients with ‘normal’ FEV₁ levels. Patients from thetwo study groups with a FEV<80% and without a primary diagnosis ofbronchiectasis, were defined as having COPD and are the subject of thisanalysis. The analysis examines the treatment with HI-164OV in all COPDpatients and then specifically treatment in the moderate to severe COPDpatient group.

All subjects were followed for 8-9 months, over the southern winter andearly spring: Recruitment began 7 Mar. 2006 and the last patientcompleted 31 Oct. 2006. HI-164OV tablets contained 45 mg (equivalent ofCFU 10¹¹ bacteria) of formalin-inactivated HI-164, provided as entericcoated tablets; placebo tablets containing excipients only, wereprovided as enteric-coated tablets for oral administration. Patientstook 3 courses of tablets. Each course consisted of 2 tablets daily for3 consecutive days (before breakfast), with courses repeated at day 28and day 56. Subjects had face-face interviews at weeks 0, 4, 8, 12, 18,24, 30, 36. At weeks 4 and 8 the next tablet course was given. At allvisits acute episodes and their characteristics were documented with theaid of a structured questionnaire; adverse events were logged; sputumand blood was collected to monitor bacterial colonization and safetyparameters respectively; and concomitant medications assessed.

The primary variables assessed for this analysis were (i) the number ofexacerbations (defined as ‘an increase in volume and purulence ofsputum’); (ii) the proportion of subjects experiencing one or moreexacerbations; (iii) the mean duration of exacerbations; (iv) the numberof antibiotic courses prescribed for exacerbations. The secondaryvariables assessed were adverse effects (including hospitalisation), andbacteriology of sputum.

Statistical analysis on the intention-to-treat population used ‘Pvalues’ considered as two-sided. Rates of acute episodes (or ‘acuteepisodes defined by treatment’) were calculated as number of eventsdivided by exposure (calculated as number of days from randomizationuntil last visit) multiplied by 100. Repeated measures Poissonregression was used to examine treatment differences in rates, where thelogarithm of exposure (in days) was considered an offset. Rate (risk)ratios and their associated 95% confidence intervals (CI) were obtainedfrom these regressions. Fisher's exact test was used to compare theproportions of subjects with one or more episodes. Durations of episodes(start date to stop date) were estimated via the Kaplan-Meier procedureand treatment differences were analyzed by the log-rank test. Forcalculation of treatment days, the number of days was calculated as thesum of duration of each pertinent medication regardless of overlap interms of start and stop dates. The number of days each subject receivedtreatment was compared between treatments using Wilcoxon's rank-sum test(student's ‘t’ approximation). The number of subjects experiencing 0, 1,2 etc. antibiotic courses was presented and treatment groups werecompared using the Cocharan-Mantel-Haenszel test. Hospitalization rateswere compared between treatments using Poisson regression, where thelog-transformed number of subjects was used as an offset.

The aim was to recruit 124 in each of the moderate-severe and themild-moderate groups, allowing for a 20% drop out rate. With 50 in eachgroup completing the study this would (at the 5% significance level andwith 80% power) detect a relative risk reduction in number of episodesof 37% (assuming an infection rate in the placebo group of 2); areduction in the proportion of patients at 28% (assuming 80% of theplacebo group have at least one infection); a relative risk reduction ofprescribing antibiotics of 33% (assuming a prescription rate of 2.4 inthe placebo group); a difference in mean days of duration of 1.2assuming a SD of 2 days in the placebo group. The requirement of 2 ormore episodes per year for 2 years proved a major impediment torecruitment, with a total of 73 being enrolled with COPD (38 withmoderate-severe disease and 35 with mild-moderate disease). Anadditional 67 subjects with bronchiectasis or with ‘normal’ FEV₁ valuesas a percentage of predicted normal (i.e., 80%) were studied, but arenot included in this analysis of those with COPD. Pooled analysis was apre-study decision.

1.15.1 Demographic Characteristics

The patient characteristics are set forth in Table 6 below.

TABLE 6 Patient characteristics All Subjects Subjects with moderate-with COPD severe COPD Placebo HI-164OV Placebo HI-164OV Characteristic(37) (36) (20) (18) Age (years) 68.8 62.8 67.8 69.5 (median, range)(41-88) (47-87) (55-88) (47-87) Sex 25:12 25:11 14:6 15:3 (male/female)Ever-smoked 30 31 15 15 Current smoker  6  7  4  2 (median, range) FEV₁(l/sec) 1.17 1.32 0.89 1.0 (median, range) (0.4-3.0) (0.5-2.6) (0.4-2.0)(0.5-1.5)

At baseline in those with moderate-severe COPD, in the active group and13 in the placebo group had a history of ischaemic heart disease, 6 inthe active group and 2 in the placebo group had a past history of‘asthma’, 2 in the active group and 4 in the placebo group had anassociated bronchiectasis. In this group relevant therapy at baselinewas: 12 subjects in each group took salbutamol; 14 in the active groupand 13 in the placebo group took tiotropium; and 10 in the active groupand 9 in the placebo group received salmeterol/fluticosone (Seretide®).Prednisone was taken by 6 in the active group and 7 in the placebogroup. Oxygen therapy was used by 5 in the active group and 2 in theplacebo group. Approximately half of both placebo and active groups hadpre-seasonal influenza vaccination, while two thirds had hadpneumococcal vaccination.

1.16 Results

The results of HI-164OV administration vs. placebo are set forth inTable 7 below:

TABLE 7 Summary of results Total COPD Population Moderate-Severe COPDHI- Protection HI- Protection 164OV Placebo (%) (P 164OV Placebo (%) (P(36) (37) value) (18) (20) value) Number of 36 (24) 50 (25) 24 (0.19) 22(13) 29 (13) 17 (0.52) acute episodes 28 (20) 38 (23) 24 (0.26) 15 (11)23 (13) 27 (0.33) (subject 9 (10) 21 (15) 50 (0.06) 5 (4) 15 (10) 62(0.05) number) Total Antibiotic- treated Corticosteroid- treatedAntibiotic 37 (20) 70 (23) 46 (0.03) 19 (11) 48 (13) 56 (0.03) Treatment284 (20) 735 (23) 72 (0.01) 136 (11) 543 (13) 60 (0.01) courses totaldays (subject number) Oral and Acute 127 (9) 344 (16) 56 (0.26) 61 (4)289 (11) 75 (0.26) Corticosteroid Treatment days (subject number)Duration of 14.7 (3-81) 17.3 (3-74) 24 (0.19) 14.3 (3-84) 22.7 (4-74) 37(0.01) episodes mean days (range)

1.16.1 Number Of Acute Episodes

Using the protocol definition of an acute episode as “an increase involume and purulence of sputum”, for the total COPD group there were 36episodes in the HI-164OV group and 50 episodes in the placebo group,which when analyzed by monthly periods had a risk ratio of 0.76 (95% CI0.50, 1.15; P=0.19). When analyzed over three monthly periods, at a rateof events/100 days, there was a greater benefit in the active group inthe second 3 month period (Placebo: events/100 days-0.82 (first 3months), 0.74 (second 3 months); Active: 0.72 and 0.48. P value. Periodeffect 0.68, and treatment effect 0.19). Using the protocol definitionof acute episode (“increase in volume and purulence of sputum”) in themoderate-severe COPD group there were 22 episodes in the active groupcompared to 29 episodes in the placebo group, with a risk ratio of 0.84(95% CI: 0.48, 1.49), which was not significant. However, analysis bythree monthly periods, the event rate/100 days was the same for activeand placebo groups at 0.82 for the first 3 months, but reduced in theactive group compared to placebo in the second 3 months (0.6 versus0.85), representing a 29% reduction in incidence. In those withmoderate-severe disease there was almost twice as many patients (11subjects) in the placebo group with 2-4 episodes when compared to theactive treatment group (6 subjects), consistent with an effect oftherapy on reduction of recurrence. There was no or little difference inprotection in the total COPD group, but a higher level of protection(27%) was noted in those with more severe disease.

1.16.2 Proportion of Patients with Acute Episodes

The proportion of patients with acute episodes was reduced by a smallamount with therapy, except when only the more severe episodes werecaptured using the definition of ‘corticosteroid-treated’ episodes.

1.16.3 Duration of Episodes

The mean duration of episodes was less in active compared to placebogroups COPD′: 14.7 compared to 17.3 days; moderate-severe COPD 14.3compared to 22.7 days), but this difference reached statisticalsignificance only in the moderate-severe group (P=0.01).

1.16.4 Medication Usage

A significant reduction in both the number of prescribed courses ofantibiotics and the number of days of antibiotic treatment (forexacerbations) followed active treatment in both the total group ofsubjects with COPD and those with moderate-severe COPD. Protection wasstatistically significant in both ‘all COPD’ and moderate-severe COPDpatient groups, for both forms of analysis. Fewer patients receivedthree or more courses of antibiotics in the active group than in theplacebo group (for ‘all COPD’, 5 versus 14 patients' for moderate-severeCOPD, 2 versus 11 patients), supporting protection against recurrentepisodes.

Subjects that had moderate to severe COPD and were treated with the oralvaccine, were found to have a 50% reduction in the usage ofbronchodilator therapies in the active group. Eosinophil countsfollowing the administration of triple course of oral NTHi therapy werefound to have significantly reduced in only the active group. Inconclusion, the oral NTHi therapy reduces the usage of bronchodilatortherapies in acute episodes and also reduces eosinophil counts which areconnected allergic reactions specific to NTHi.

1.16.5 Admission to a Hospital

The rate of hospital admission for exacerbation of COPD was high (seeTable 8 below), with 10 admissions in 7 patients in the placebo group(20 subjects) in moderate-severe COPD. For those with less severe COPD(17 subjects) only two were admitted to hospital for an exacerbation ofCOPD through the period of the study. Protection against admission tohospital due to an exacerbation of COPD in ‘all COPD’ was 61% and ofborderline significance (P=0.07) while in those with moderate-severeCOPD, a higher level of protection was found (90%) which was significant(P=0.01). In this more severe group, a significant difference in‘hospitalisation for all causes’ was detected (P=0.04), but numbers weretoo small to identify an independent effect on hospital admission fornon-respiratory events.

1.16.6 Sputum Bacteriology

Analysis of ‘acute event’ sputum samples gave information of littlevalue due to antibiotic treatment, transport delays and latenotifications. Specimens collected at regular visits following the onsetof the study were considered valid if cultures were made less than 4hours from collection, and saliva contaminated specimens (detected bypresence of epithelial cells) were excluded. For these ‘routine’ samplesin the placebo group for ‘all COPD’ (37 subjects), half the patients (19subjects) had positive cultures with a total of 88 positive cultures.The equivalent figures for the active group (36 subjects) were 13subjects with 45 positive cultures, i.e., following HI-164OV therapythere were less subjects (by 31%) having positive sputum cultures forrespiratory pathogens, with the total number of positive sputum culturesbeing approximately half that in the placebo group. In the placebo groupNTHi was isolated in 14 subjects on an average of 2.8 occasions. Forother pathogens: M. catarrhalis was isolated in 5 subjects (average 2.4occasions); S. pneumoniae in 6 subjects (average 1.7 occasions); P.aeruginosa in 11 subjects (average 2.3 occasions). In the active groupNTHi was isolated in 8 subjects (average 2.9 occasions); M. catarrhalisin 7 subjects (average 1.0 occasion); S. pneumoniae in 4 subjects(average 1.5 occasions); P. aeruginosa in 3 subjects (average 3occasions). Further analysis showed in the placebo group the 21 subjectswith positive sputum cultures had 34 acute episodes (1.62 per subject)while the 16 without positive samples had 16 acute episodes (1.0 persubject). For the active group, 14 with positive cultures had 20 acuteepisodes (1.42 per subject) and 22 with a positive sputum culture had 16acute episodes (0.73 per subject). For NTHi isolates, a similar patternwas shown (placebo: positive culture in 14 subjects with 20 acuteepisodes (1.42 per subject) and no growth in 23 who had 30 acuteepisodes (1.31 per subject); active: 8 subjects with positive cultureshad 10 episodes (1.25 per subject) while 28 with no growth had 26episodes (0.93 per subject). It is concluded (i) that a positive sputumculture predicts a greater risk of having an acute clinical episode(irrespective of whether the subject is in the placebo or active group),(ii) that in the active group, there is apparent protection in bothculture positive and culture negative groups (approximately 12% in theculture positive group—calculated as infection episode/person value of1.42 in the active versus 1.62 in placebo group; and approximately 27%in the ‘non culture positive’ group—calculated as infectionepisode/person value of 0.73 in the active versus 1.0 in placebo group),(iii) HI-164OV oral therapy is followed by less of positive cultures.These ‘no growth’ cultures are associated with less exacerbations(above). A similar analysis of NTHi positive sputa gave almost identicaloutcomes with respect to the protective effect of oral HI-164OV therapy(data not shown).

TABLE 8 Hospitalization HI-164OV Placebo Number Number Number Number ofof of of P value events patients events patients 1 2 (a) All COPD AllHospitalization 8 8 16 12 0.12 0.50 events Hospitalization for 5 5 13 90.07 0.48 exacerbations of COPD (b) Moderate- severe COPD AllHospitalization 3 3 12 8 0.04 0.53 events Hospitalization for 1 1 10 70.01 0.87 exacerbation of COPD P(1) compares hospitalization rates inall subjects. P(2) compares hospitalization rates in hospitalizedsubjects.

TABLE 9 Protection in active versus placebo in sputum negative subjectsNo. No. subjects subjects with with sputum sputum samples samples (no.acute (no. acute episodes) episodes) Active Placebo Any pathogen + 21(34) 14 (20) 1.42 1.62 12 grown in − 16 (16) 22 (16) 0.73 1.0 27 routinesputum samples Non-typeable + 14 (20)  8 (10) 1.25 1.42 12 Haemophilus −23 (30) 28 (26) 0.93 1.31 29 influenzae grown in routine sputum samples

TABLE 10 Protection in active versus placebo in sputum negative subjectsOrganisms Percentage of Percentage of Clinical isolated from Subjects(%) Protection (%) sputum HI-164OV Placebo culture +ve culture −ve Anypathogen 36 51 12 27 grown in routine sputum samples Non-typeable 22 3812 29 Haemophilus influenzae grown in routine sputum samples

Tables 9 and 10 demonstrate the percentage of bacteria grown fromvarious sputum samples of the subjects/patients. The Active Group wastreated with HI164OV product.

1.17 Discussion

Acute exacerbations as key events in the natural history of COPD arerecognized as particular targets for intervention therapy in currentguidelines. To determine the impact of immunotherapy with an oralvaccine (HI-164OV) prepared from a novel isolate of NTHi (NTHI-164) onthe incidence and severity of exacerbations, subjects with COPD who hada history of recurrent exacerbations were studies. Such subjectshowever, represent only 20-25% of all those with COPD, which led in thepresent study to an enrolment less than was planned. This difficulty wasfurther complicated by the use of a classical descriptive definition ofacute episodes as ‘an increase in volume and purulence of sputum’. Whilethis definition reflects the infective origin of acute episodes, it canbe imprecise given the variable background level of intrabronchialinflammation in COPD, blurring distinction of discrete episodes. Despitelimitations imposed by definitions, two conclusions can be taken fromthis study. First, subjects with COPD and recurrent exacerbationsbenefit from oral immunotherapy with HI-164OV, in particular with areduction in their severity. Second, those with moderate-severe COPDbenefit to a greater extent than do those with milder disease.

Although there was a trend to a reduction in incidence of exacerbationsin ‘all COPD’ using the descriptive definition of increase in sputumpurulence, several observations support this outcome measure as havingclinical relevance. Analysis in the placebo group showed a high eventrate at the beginning of the study. A higher protection level at 35% inthe second three months of this study is therefore consistent with adelayed onset of immune enhancement, with results in the second half ofthe study a more accurate reflection of protection. Moreover, theplacebo group included more than twice the number of subjects having 2-4episodes, suggesting that HI-164OV treatment is particularly effectivein preventing recurrent episodes. Persuasive data come from the analysisof episodes defined in terms of treatment, especially withcorticosteroids, when there was a protection level in excess of 50%.These data sets showing clearer cut protection for more severe episodes,indicate that the main value of immunotherapy relates to reduction inparameters of exacerbation severity, at least in those with COPD andfrequent episodes.

There was a difference in parameters of severity following activetreatment with a consistency of benefit in all parameters of severity,with HI-164OV treatment followed by shorter episodes, less treatmentwith antibiotics and oral corticosteroids, and less chance ofhospitalization. Usage of antibiotics in terms of courses prescribed, ordays taken, was an improved index of benefit rather than simplyanalyzing number of episodes treated with antibiotics, suggesting thatmost episodes of purulent sputum will be treated with an antibioticirrespective of severity, but only those considered to be clinicallysevere will attract repeat courses. The significant reduction inhospitalization in those with moderate-severe disease is an impressiveoutcome given the small sample size. Applicants are unaware of any otherintervention therapy having a similar ability to reduce hospitalizationrate. The numbers are small, and this outcome was only significant inthe group with moderate-severe COPD, but this is consistent withimprovement in all other parameters of severity having the greatestimpact in those with moderate-severe disease. This may reflect a greaterclinical benefit for any given reduction in airways inflammation, inthose with the most compromised airways.

The standard for comparison of therapy aimed at reducing the impact ofacute exacerbations in COPD is fluticasone/salmeterol (500/50 mcg).Studies of this combination included large numbers of subjects withprimary outcomes measuring retention of flow rates or all-causemortality. Subjects included in the studies had moderate-severe COPD,but were not selected because of frequent acute episodes as in thepresent study. A protection level of about 40% against acute episodesdefined by corticosteroid-therapy was observed in each study, less thanin the HI-164OV study but with significance levels reflecting thegreater power possible with large numbers. Benefit was particularlynoted in those with more severe disease, as found in the HI-164OV study.However, fluticasone/salmeterol protected little if at all (0% and 17%)against ‘severe’ exacerbations defined as requiring admission tohospital, in contrast to the data obtained using HI-164OV. It is notedthat in this study that the hospitalization rate of about 9% per annum,compared to a higher rate in the present study by two to three fold.This higher rate probably reflects frequent exacerbations as a marker ofmore severe disease. When acute episodes were defined in terms ofantibiotic-therapy, however, there was an increase in number of episodesin the active group. This contrasted with a reduction ofantibiotic-treated episodes in the HI-164OV study. The increase inantibiotic-defined acute episodes is consistent with an inhaledsteroid-related proneness to infection (oropharyngeal thrush andpneumonia).

Early longitudinal studies of patients with COPD failed to clarify therole played by bacteria in pathogenesis mainly because the rate ofisolation of bacteria in sputum during stable disease was similar tothat during acute episodes. Recent prospective studies have usedmolecular typing to demonstrate an association between new strains ofbacterial pathogens with exacerbations of COPD. Isolation frequencies ofbacteria in this latter study, in routine sputum samples, were verysimilar to those found in the present study, and the isolation of apathogen was significantly associated with a current exacerbation. Thetrend in the data from the present studies obtained in stable disease,is consistent with these concepts: positive sputum culture appeared tobe a risk factor for acute exacerbations (irrespective of whether thesubject was in the placebo or active limb of the study); and comparingactive with placebo, added protection is noted in the active group inboth those growing pathogens and those who do not. These outcomes couldreflect enhanced mucosal resistance to acquisition of ‘novel’ bacterialstrains, and/or to a reduction in the level of bacteria colonization ofthe airways, that resists pro-inflammatory events such an intercurrentvirus infection. In either situation a specific activation of Peyer'spatch-derived T cells by NTHi antigens mediates a recruitment andactivation of phagocytic cells which serve as a non specific effectormechanism.

Oral immunotherapy with killed NTHi (HI-164OV vaccine) activates theGut-Associated Lymphoid Tissue (GALT), with specific T lymphocyteshoming to the bronchus mucosa. Recent studies have shown that aspirationof bronchus contents into the gut is a physiological mechanism ofantigen presentation to the GALT, and that oral administration ofHI-164OV serves to optimize a seasonal process of T cell activation.HI-164OV is a different product to the polybacterial extracts usedwidely in Europe for ‘immune protection’. It contains a single speciesof bacteria, formulated as a whole cell preparation at a concentrationseveral logs greater than those used in the polybacterial products, soas to drive a specific Peyer's patch immune response. Analysis ofstudies using polybacterial isolates in subjects with chronic airwaysdisease failed to show significant reduction in exacerbations, anoutcome also found in a comparative study between a polybacterialproduct and oral NTHi immunotherapy.

Example 4: NTHi-164 Conveys a Greater Degree of Cross-Protection thanNTHi-166 in Challenge Experiments

NTHI-164 and NTHI-166 (NTHi289) tested as vaccines in a rat model ofintra-tracheal (IT) immunization and acute respiratory infectionchallenge with a panel of NTHi isolates gave different levels ofprotection. The isolates used in the infection challenge were NTHI-164,NTHi-165, NTHI-166, NTHI-167, NTHi-168, ATCC 3041, ATCC 43095, ATCC35092 and ATCC51997.

To determine the protection provided by the vaccines groups of DarkAgouti rats (5-6 rats per group) aged 8-10 weeks old were immunizedintra-tracheally (IT) with 50 uL of PBS or with 50 uL of PBS containing5×10⁸ killed NTHI-164 or NTHI-166, on days 0 and 14. On day 21 rats wereinfected with one of a panel of infection challenge NTHi strains. Therats were killed after 4 h of infection and lungs lavaged with 5×2 mLPBS to give 10 mL of broncho-alveolar lavage (BAL) fluid. The lungs werethen homogenized in 10 mL PBS top give a lung homogenate (LH)suspension. Twenty microliter samples of serial 10-fold dilutions of BALand LH were plated out on chocolate agar and incubated overnight at 37°C. in 5% CO₂ in air. The colonies were counted and the number of liveNTHi in BAL and LH calculated for each rat. The levels of total bacteriain the lung were also determined for each rat by addition of bacterianumber in BAL and LH. The mean bacteria level was calculated for eachgroup of rats and the % bacterial clearance determined compared to thecontrol PBS group as follows:

$\frac{\begin{matrix}\left( {{{Mean}\mspace{14mu} {bacteria}{\mspace{11mu} \;}{in}\mspace{14mu} {PBS}{\mspace{11mu} \;}{group}} -} \right. \\{\left. {{Mean}\mspace{14mu} {bacteria}{\mspace{11mu} \;}{in}\mspace{14mu} {vaccine}{\mspace{11mu} \;}{group}} \right) \times 100}\end{matrix}}{{Mean}\mspace{14mu} {bacteria}\mspace{14mu} {in}\mspace{14mu} {PBS}\mspace{14mu} {group}}$

The percentage bacterial clearance in 4 hours is a measure of the degreeof protection provided by the vaccine.

The mean percentage bacteria clearance provided by each test vaccine forthe panel of 9 challenge infection strains was calculated. For theNTHI-164 vaccine the mean bacterial clearance for the panel was 84±4%,and for the NTHI-166 vaccine the mean bacteria clearance for the panelwas 78±2%. This demonstrates that a vaccine prepared from killedNTHI-164 gives a greater degree of bacterial clearance against the panelof infecting strains than does a vaccine prepared from NTHI-166 and isthus more efficacious as a whole-cell vaccine.

Example 5: HI-164 Elicits a Non-Specific Immune Response AgainstNon-Haemophilus Species

A multi-centre, double blind, placebo controlled, prospective study toassess safety and efficacy of orally administered killed whole cellnon-typeable Haemophilus influenzae (NTHi) vaccine containingformalin-killed NTHI-164 (herein referred to as “HI-164OV”) inpreventing episodes of acute bronchitis in patients with moderate tosevere airway disease.

1.18 Objectives & Methods

1.18.1 Primary Objectives (in Active Vs. Placebo Groups):

-   -   Compare number of acute bronchitis episodes    -   Compare proportion of patients with an episode of acute        bronchitis    -   Compare duration of episodes of acute bronchitis

1.18.2 Methodology

Prospective, double blind, placebo controlled, parallel group,multi-centre study to assess the safety and efficacy of orallyadministered killed whole cell NTHi HI-164OV in preventing episodes ofacute bronchitis in patients with moderate to severe (FEV₁≤50% of thepredicted value) airway disease.

Patients were randomly allocated in a 1:1 ratio to either studymedication or placebo. Patients took medication on 3 consecutive dayseach month for 3 consecutive months. Microbiology evaluations wereconducted on throat gargles for quantitative detection of Hi, and asputum sample for Gram stain and quantitative detection of Hi andsemi-quantitative detection of M. catarrhalis, S. pneumoniae andPseudomonas species.

1.18.3 Number of Patients (Planned and Analyzed)

The study sample size was calculated on the basis of a 5% significancelevel and 80% power, a sample size of 50 participants per group wouldallow detection of:

-   -   A relative risk for infection of approximately 0.63 (i.e.,        relative risk reduction of 37%) assuming infection rate in the        placebo group of 2;    -   An absolute reduction in the proportion of patients experiencing        any infection of 28% (assuming 80% of placebo groups have any        infection);    -   A relative risk for antibiotic prescribing of 0.67 (i.e.,        relative risk reduction of 33%) assuming a prescription rate of        2.4 in the placebo group;    -   A difference in mean days of duration of infections of 1.2 days        (assuming standard deviation of duration of 2 days).

Assuming that 20% of participants did not complete the study, 62 neededto be recruited for each group with 124 to be randomized. Actual numberrandomized and analyzed=38. There were two dropouts in each treatmentgroup, with one in each group discontinuing the study due to an adverseevent. Patients were recruited at 4 study centers. Note patientrecruitment did not reach the planned number due to the limitedavailable population with the required severity of illness who also metthe inclusion criterion of 2 episodes of acute bronchitis per year inthe previous 2 years at the 4 participating centers.

1.18.4 Product, Dose, & Batch Number

HI-164OV enteric-coated tablets: Contained 45 mg of formalin-inactivatedNTHi-164 plus excipients. The lyophilized active substance is referredto as HI-164. Placebo enteric-coated tablets contained only excipients.Placebo and HI-164OV were stored at 2-8° C. and tablets weighed about450 mg.

1.18.5 Administration & Study Duration

Patients took study medication orally with a glass of water, on an emptystomach, 30 minutes before breakfast on 3 consecutive days each monthfor 3 consecutive months (days 1-3, days 29-31 and days 57-59). Studyduration was approximately 8 months.

Patients took study medication on 3 consecutive days each month for 3consecutive months (days 1-3, days 29-31 and days 57-59). Study durationwas approximately 8 months.

1.18.6 Criteria for Evaluation

The primary efficacy variables were:

-   -   Number of episodes of acute bronchitis    -   Proportion of patients on treatment experiencing an episode of        acute bronchitis    -   The duration of acute bronchitis episodes    -   The number of courses of antibiotics for the treatment of acute        episodes of bronchitis    -   The presence of H. influenzae, M. catarrhalis, S. pneumoniae and        Pseudomonas spp in sputum    -   Severity of episodes of acute bronchitis

1.18.7 Statistical methods

Statistical analyses were performed on the intention-to-treat populationof 38 patients, namely all patients who were randomized to treatmentregardless of whether they took study drug. All relevant data weresummarized by treatment group. The analysis consisted of three sections:

-   -   Recruitment, and status at the end of the study    -   Baseline characteristics    -   Evaluation of treatment effects on the primary and secondary        objectives.

The primary hypothesis tested was the superiority of HI-164OV overplacebo in preventing episodes of acute bronchitis. The numbers ofepisodes of acute bronchitis were compared between groups using repeatedmeasures GEE Poisson regression and the Cochran-Mantel Haenszel test.The proportion of participants in each treatment group who developed oneor more episodes of acute bronchitis within the trial follow-up periodwere compared using Fisher's Exact Test. The duration of the acutebronchitic episodes was compared using the log-rank test and Wilcoxonrank-sum test.

Acute bronchitic episodes were adjudicated on the investigator reportsand the adverse events which were of a respiratory infective nature;verification was obtained from the study site where needed. The numberof courses of antibiotics for the treatment of the episodes of acutebronchitis was compared using the Cochran-Mantel Haenszel test. Thetotal duration (number of days) of courses of antibiotics for thetreatment of the acute episodes was compared using the Wilcoxon rank-sumtest.

1.18.8 Patient Population Summary

The recruitment target of 124 randomized patients was not met; theactual number of patients screened was 42 of whom 38 were randomized and34 completed the study. Of the patients randomized, there were 2patients in each group who did not complete the study, of whom onepatient in each group discontinued because of an adverse event. Baselinepatient demographics were generally similar for the HI-164OV and placebogroups. The mean age of the population was about 70 years. More malesthan females were enrolled with 83.3% and 70.0% males and 16.7% and30.0% females for the active and placebo groups, respectively. The meanFEV₁ in all patients was 0.95 litres/sec and similar between active andplacebo groups; the median values were 1.00 and 0.85 in the active &placebo groups, respectively. A larger proportion of patients in bothgroups reported a history of smoking compared with never having smoked(84.2% smoking versus 15.8% never smoked). Patients had smoked for amean of 51.36 pack years and a median of 45.5 pack years; with 45.0 &46.0 median pack years for the active and placebo groups, respectively.At screening, 15.8% of all patients (with a similar distribution betweenthe groups) still smoked.

Medical history was consistent with a population of patients of thisage, the majority of whom smoked and had recognized airways disease.Significantly more patients reported a history of cardiac disorders inthe placebo group compared with the HI-164OV group (13 (65%) vs. 5(27.8%); p=0.028). There were no other statistically significantdifferences in the distribution of conditions between the groupsaccording to system organ classes. With regards to respiratorydisorders, COPD was reported 61.1% and 45.0% in the active and placebogroups, respectively; asthma reported in 33.3% and 10.0% in the activeand placebo groups, respectively; bronchiectasis reported in 11.1% and20.0% in the active and placebo groups, respectively; and emphysemareported in 22.2% and 35.0% in the active and placebo groups,respectively. Infections and infestations were reported in 8 (44.4%) ofthe HI-164OV group and 10 (50%) of the placebo group.

1.18.9 Primary Efficacy Variables

Number of episodes of acute bronchitis: There were 22 episodes in 13 of18 patients in the HI-164OV group and 29 episodes in 13 of 20 patientsin the placebo group. The analysis of acute bronchitic episodes usingmonthly periods, showed a risk ratio of 0.83 [95% CI 0.47, 1.46],indicating a benefit for treatment but not achieving statisticalsignificance (p=0.520). Analysis using three-monthly periods showed thatthe risk ratio was 0.84 [95% CI 0.48, 1.49], also indicating a benefitof treatment which was not statistically significant (p=0.397). However,whilst there was a similar rate of acute bronchitic events in the first3 months of the study (0.82 events/100 patient days in each group), inthe final months the rate was 0.85 in the placebo group but 0.60 in theHI-164OV group. This suggests a benefit of HI-164OV in this secondperiod after all three cycles of drug had been administered althoughthis was not significant (regression p values: period 0.894, treatment0.397, interaction 0.533). The reduction of approximately 30% in thenumber of episodes in the second period is clinically relevant as thestudy began late in the autumn and a lag period of 6-8 weeks beforebenefit manifests is suspected from earlier studies.

When analyzed according to the period March-May versus the rest of thewinter season, the treatment effect (risk ratio) was 0.92 [95% CI 0.50,1.67], again suggesting an overall benefit of treatment with HI-164OValthough this was not statistically significant (p=0.262). The rate ofevents in the groups during these periods was 0.69 events/100 patientdays in the placebo group and 0.85 events/100 patient days in theHI-164OV group in March-May. In June-October the rate was 0.90events/100 patient days in the placebo group and 0.59 events/100 patientdays in the HI-164OV group.

Proportion of Patients on Treatment Experiencing an Episode of AcuteBronchitis:

There was no statistically significant difference in the proportion ofpatients experiencing an episode of acute bronchitis in the two group(p=0.734).

The Duration of Acute Bronchitis Episodes:

The median duration of acute bronchitic episodes in the HI-164OV group(8.5 days) was shorter compared with placebo (15.0 days), however, thiswas not statistically significant different (p=0.0879). However, therewas a significant difference in the mean duration of episodes:14.32±17.01 (SD) days in the HI-164OV group and 22.72±18.76 (SD) days inthe placebo group (p=0.0141).

Analysis of the median time to the first episode of acute bronchitis wasnot statistically significantly different (81.0 days in the HI-164OVgroup and 100.0 days in the placebo group; p=0.7962).

The Number of Courses of Antibiotics for the Treatment of Acute Episodesof Bronchitis:

The number of patients treated with antibiotics for acute bronchiticepisodes was similar between the HI-164OV and placebo groups (11 versus13 respectively). In these same patients, there were fewer courses ofantibiotics prescribed in the HI-164OV group (19 courses versus 48 forplacebo); this difference was significant, p=0.031. (This calculationwas based on a retrospective clinical review of the antibioticsprescribed for acute episodes of bronchitis). In addition, there weremore patients with more courses of antibiotics in the placebo group (5patients with 3 courses and 6 with ≥4 courses) than in the active group(1 patient with 3 courses and 1 with ≥4 courses). There was >75%reduction in the total number of days of antibiotic treatment for theacute bronchitic episodes; a total of 543 days in the placebo group and136 in the HI-164OV group (p=0.013). The mean numbers of days ofantibiotic therapy administered per patient was 41.77 days (range 5-117days) in the placebo group and 12.36 days (range 1-41 days) in theHI-164OV group.

1.18.10 Secondary Efficacy Variables

Measure of surrogate mucosal immune markers of protection (NTHi-specificantibody in serum): Antibody to NTHi antigen was detected at thebeginning and end of this example study. No significant difference wasnoted between placebo and active groups for either the mean levels orthe degree of variation (determined by large SE bars) as had been notedin the HI-H003 study of subjects who smoked but most of whom wereunaware of airways disease reflecting different levels of intrabronchialcolonization. In those with moderate to severe COPD (HI-H002 study)there was a small but significant difference in the change in IgG antiNTHi antibody (0.15 of a log₁₀; p=0.004) with a trend of a similardirection of a change for IgA anti NTHi antibody (0.09 of a log₁₀;p=0.08). This significant treatment effect was not seen in those with anFEV₁ of >50% predicted. The mechanism is unclear. More frequentmonitoring of the antibody response to treatment may provide a clearerpicture of mechanism of change.

The presence of H. influenzae, M. catarrhalis, S. pneumoniae &Pseudomonas spp in sputum: The bacteriology results of the sputumsamples from this study showed consistent isolation of fewer non-NTHipathogens (Moraxella catarrhalis, Streptococcus pneumoniae, andPseudomonas aeruginosa) in those taking oral NTHi therapy. For examplethe total number of positive culture samples in the HI-164OV group were34 and 66 in the placebo group of which H. influenzae was isolated 17and 22 times, respectively. The ‘other pathogen’ load was 17 isolates inthe active group and 44 in the placebo group, these other colonizingpathogens being predominantly Moraxella species and Pseudomonas species.Four patients in the HI-164OV group and 8 in the placebo group had morethan one bacterium isolated during the study. These results areconsistent with the qualitative data in the earlier Broncostat™ studies.The general reduction in pathogen isolation noted following HI-164OVtreatment is interpreted to reflect the non-specific nature of the finaleffector mechanism (i.e., phagocytosis) or the greater use ofantibiotics in the placebo group.

Severity of Episodes of Acute Bronchitis:

There were no specific parameters specified in the protocol to measurethe severity of acute bronchitic episodes. However, use of systemiccorticosteroids and other concomitant medications for management ofthese episodes and also hospitalization of patients for respiratoryevents represent measures of severity. It was found that patients in theHI-164OV group received fewer treatments with systemic corticosteroidsfor episodes of acute bronchitis (4 in the HI-164OV group and 11 in theplacebo group). There was also a marked difference in the number of daysof systemic steroid treatment, although this was not statisticallysignificant (HI-164OV group: 61 days (mean 15.25 days); placebo group:289 days (mean 26.27 days); p=0.259). In addition, hospitalization tomanage of adverse respiratory events were three times greater in theplacebo group (n=7, 10 hospitalizations) than in the HI-164OV group(n=1, 1 hospitalization); this was statistically significant (p=0.0362).

1.19 Safety Results

Adverse events: The overall proportion of patients experiencing an AE inany primary system organ class was similar (18 (100%) in the HI-164OVgroup vs. 19 (95%) in the placebo group). The majority of reported AEswere mild or moderate, and were not considered to be related to studytreatment; there was 1 patient in the HI-164OV group (patient 03 who hadan episode of moderate gastroenteritis) and 2 in the placebo group(patient 14 who had moderate rash and a hot flush immediately aftervisit 1 and advised not to take more study medication; and patient 27who had mild nausea) with events which were described as being possiblyrelated to study treatment. No events were considered definitely relatedto study treatment.

One patient in the placebo group died as a result of an infectiveexacerbation of chronic airways limitation with Pseudomonasapproximately 2 months after starting the study. The patient hadcompleted two courses of study medication, and had been withdrawn fromthe study on 25, May 2006; he died on 6, Jul. 2006. The event was notconsidered to be related to study treatment. A total of eleven patients(3 patients (16.7%) in the HI-164OV group and 8 (40.0%) in the placebogroup) reported serious adverse events (not statistically significant;p=0.160), but none were related to treatment. These eleven patients (3in the HI-164OV group vs. 8 in the placebo group) were hospitalized atotal of 15 times (3 events leading to hospitalization in the HI-164OVgroup versus 12 in the placebo group; p=0.0472). More than three timesas many patients were hospitalized for management of a respiratory eventin the placebo group (n=7, 10 hospitalizations) than in the HI-164OVgroup (n=1, 1 hospitalization); this was statistically significant(p=0.0362). The number of patients hospitalized for non-respiratoryevents was the same in both groups (2 patients).

This example study evaluated the effects of HI-164OV versus placebo asan add-on treatment in 18 and 20 patients, respectively, with moderateto severe airways disease (demonstrated by an FEV₁ of around 1.0liters/sec and ≤50% of the predicted value) who had at least 2 episodesof acute bronchitis per year in the previous 2 years. Benefit oftreatment with HI-164OV was observed in the primary endpoints, although,given the small size of the treatment groups, not all Analyses reachedstatistical significance. The higher number of patients with cardiacdisease in the placebo group is likely to have negatively affectedresponse to infection. Consistency of benefit was evident in the primaryefficacy endpoints. There was a 16% risk reduction in the number ofacute bronchitic episodes in the HI-164OV group and around a 35%reduction in the mean duration of these episodes (p=0.0141) comparedwith placebo. The effect of HI-164OV was evident in the first 3 monthsof therapy and maintained over the study. In addition, there was a 60%reduction in the courses of antibiotic employed to treat the bronchiticepisodes and a 75% reduction of total days of antibiotic therapy. Elevenpatients in the placebo group and 4 in the HI-164OV group receivedsystemic (oral or intravenous) corticosteroids to manage the acutebronchitic episodes. These results were further supported by a reductionin hospitalization for respiratory events; there was a 80% reduction inthe number of patients being hospitalized in those taking HI-164OVcompared to placebo (7 vs. 1) and a 90% reduction in the number ofhospitalizations (10 vs. 1). The rate of hospitalization for otherreasons was the same between the 2 groups. Importantly, HI-164OVdemonstrated an acceptable safety profile. There was no particularpattern of adverse events in the HI-164OV group and no overt issuesdetected in the vital signs or laboratory data.

The bacteriology data shows that there was a higher bacterialcolonization of H. influenzae and also the other pathogens in theplacebo group. This is interpreted as being related to the efficacy dueto the efficacy of HI-164OV and also either due to or in spite of thehigher use of antibiotics in the placebo group. There was a slight risein the NTHi specific IgG and IgA levels.

The resulting data depicted in FIGS. 11 and 12 demonstrate the successof the specific antigen immune response and the non-specific immunesystem stimulation by HI-164OV. FIG. 11 shows the results of allpatients (excluding sputum contamination) with respect to the number ofinfections experienced by a control group compared with the group ofpatients who had taken the recommended or suggested course of HI-164OV.These results demonstrated that there was a significant fall ininfection rates not only with H. influenzae but also in respect of otherorganisms known to infect the airways (including, but not limited to: M.catarrhalis, S. pneumoniae, Streptococcus species, P. aeruginosa,Pseudomonas species, S. aureus and/or Mycobacterium species) and mucosallayers of patients. These results particularly highlight the fact thatHI-164OV is generating both a specific and non-specific immune responsein immunized patients.

Also importantly, the results of FIGS. 11 and 12 demonstrate that thespecific and non-specific immune responses are approximately equal orbalanced. In particular, the specific response against Hi limits orprevents the approximately same amounts of infections by secondarybacterial infections (such as by M. catarrhalis, S. pneumoniae, P.aeruginosa, Pseudomonas species and/or Klebsiella pneumoniae). Pleasenote that for purposes of this specification “approximately equal” isdefined as being within a range of 0-5 fold.

Please note that the results also demonstrate that for a non-specificimmune response to occur, the patient is not required to be acutelyinfected or previously exposed with the primary bacterial antigen, whichin this embodiment is NTHi. Therefore the oral vaccine described in thisembodiment may effective in protecting or limiting the risk of infectionin patients not previously exposed to NTHi or Hi for the oral vaccine tobe successful.

The success of the results is further highlighted when the results arenarrowed to investigate of a subgroup of the patients suffering fromsevere airways diseases such as COPD. The results of this subgroup aredepicted in FIG. 12 and show even greater falls in infection rates withrespect to these patients. Again, the HI-164OV has demonstratedeffectiveness in use for generating both specific immune responses to Hiand non-specific immune responses to other bacterial organisms andthereby reducing the overall rates of infection. Please note that thenon-specific immune responses may prevent or limit other bacterialinfections of the mucosal layer or airways and fall within the scope ofthis specification.

In FIG. 13, the differences between Broncostat™ (as described in PCTApplication No. WO86/05691) and HI-164OV are readily apparent. Thistable depicts the increase in stimulation index in the blood T-cellsproliferation following the administration of the respective vaccines.Even after week 62, HI-164OV is stimulating T-cell proliferation whilstBroncostat™ fails to accomplish this in a statistically significantmanner. The data in this table demonstrate the non-specific immunesystem stimulation by a vaccine of the present application.

Example 6: Physical Characteristics of Strain HI-164

1.20 Materials and Methods

1.20.1 Preparation of Whole Bacterial Extracts

Method I—Bacteria Grown on Chocolate Agar Plates

A chocolate agar plate was inoculated with a single colony of NTHI-164or other Haemophilus influenzae isolate. The plate was incubatedovernight at 37° C.+5% CO₂. This resulted in heavy growth. The bacteriawere removed from the surface of the plate with a sterile 10 μl loop andimmersed into 0.5 mL of CelLytic B Cell Lysis Reagent in a microfugetube. (Bacteria were dislodged by rubbing the loop between two fingersto twirl it while immersed in the lysis buffer). The tube was allowed tostand for 5 minutes to allow the bacteria to lyse completely. The tubewas then centrifuged at 11,500 g for 5 minutes to pellet any insolublecell debris. The supernatant was then carefully removed into a sterilemicrofuge tube and stored at −70° C. until required. The proteinconcentration of the sample was determined by performing a ProteinEstimation Assay using the Pierce BCA protein assay kit.

Method II— Bacteria Grown in Tryptone Soya Broth+HTM Supplement

Tryptone Soya Broth base and HTM supplement were obtained from Oxoid.

To prepare the Tryptone Soya Broth with HTM supplement 12.5 gm of TSBpowder was weighed out and place into 1 L Schott Bottle. Then 2.5 g ofyeast extract was weighed out and added to the bottle. Distilled water(0.5 L) was added and the bottle shaken to dissolve the materials. Themedium was then autoclaved for 15 minutes. The medium was allowed tocool overnight to room temperature. On day two, using sterile technique,2 mLs of sterile distilled water was added to the vial of HTM Supplementusing a 3 ml syringe and 19G needle. This was swirled to dissolve thesolids and was stood at room temperature for 30 minutes. The contents ofthe vial were added to the bottle of medium using a sterile syringe andneedle. The contents of the Schott bottle were mixed and labeled withthe date. The medium was stored at 2-8° C. for up to 1 month. Achocolate agar plate was inoculated with a single colony of HI-164. andincubated overnight at 37° C.+5% CO₂. (This resulted in heavy growth).On day 3 100 mLs of the prepared TSB+HTM medium was measures into a 250mL Erlenmeyer conical flask and pre-warmed in a 37° C. shakingincubator. A preparation of live NTHI-164 at 1×10¹⁰ was prepared byharvesting the bacteria from the plate into PBS, measuring the opticaldensity, comparing to a previously prepared plot of optical densityversus bacteria concentration and adjusting the concentration asrequired. The broth was inoculated with 1.0×10¹⁰ live NTHI-164 (1 mL of10¹⁰/mL) to give a starting concentration of 1×10⁸/mL and mixed byswirling. The bacterial suspension was incubated in the 37° C. shakingincubator for 11 hrs overnight. By this point the bacteria were at anoptimal growth phase and expressing proteins of interest. To determinethis an OD reading at 595 nm of the suspension was determined every halfhour. When the OD had become stable, the growth curve had reached thestationary phase. 100 μl of the suspension was pipetted into a well of aflat bottom 96 well microtitre plate and read at a wavelength of 595 nm.The optical density was required to be between 0.5-1.0. If thesuspension was higher than 1.0 it was adjusted by adding extra sterilebroth to the flask. When adjusted 1.5 mL of the bacterial suspension wascentrifuge at 11,500 g for 5 minutes. The supernatant was removed andthe pellet re-suspended in 0.4 mL of CelLytic B. This was brieflyvortexed and mixed for 5-10 minutes to ensure full extraction of thesoluble proteins and was centrifuged again at 11,500 g for 5 minutes topellet any insoluble cell debris. The supernatant was carefully removedinto a sterile microfuge tube and stored at −70° C. until required. Theprotein concentration of the sample was determined by performing aProtein Estimation using the Pierce BCA protein assay kit.

1.20.2 Single Dimension Polyacrylamide Gel Electrophoresis

Twenty micrograms of protein from each extract was diluted to 20 uL withdouble-distilled water, and 10 uL of SDS-Stop solution was added.Samples were loaded into the wells of a 12% SDS-PAGE gel along withBIORAD precision molecular weight markers. Samples were run at 200Vuntil the dye front reached the bottom of the gel. Gels were thenstained with Sigma EZBlue stain.

1.20.3 Two-Dimensional Polyacrylamide Gel Electrophoresis

100 μg of each sample was acetone precipitated and resuspended inrehydration buffer. 7 cm, 3-10 NL IPG strips were used for the 1^(st)dimension, with a total focusing time of 5 hours and 18750 Vhrs. IPGstrips were loaded into the top of 12% i.5 mm thick SDS-PAGE gels andsealed with agarose. Molecular Weight Agarose plugs were also added tothe top of the gels. Gels were run for 10 mins at 50V and then 45 minsat 200V. Gels were stained with Coomassie Blue and destained for 24 hrs.Gels were then scanned and spots detected.

1.20.4 MALDI Analysis of Proteins

Protein band or spots were excised from the SDS-PAGE gel and placed in1.5 ml tubes. Bands were destained using 3 changes of 50% methanol/50 mMAmmonium Bicarbonate and air-dried overnight. Bands were then rehydratedusing 200-400 ng of Trypsin dissolved in 20 mM Ammonium bicarbonate.After 10 minutes a further 5-10 μl of 20 mM ammonium bicarbonate wereadded to each band if necessary. Bands were incubated at 37° C. for 3hours. Bands were then macerated and 1 μl samples removed. Samples werethen mixed 1:1 with CHCA matrix and spotted onto a MALDI slide.

Samples were then analyzed using an Amersham ETTAN Maldi-ToF to generatea peptide mass fingerprint. Matches were searched for using the “nr”database as part of the analysis. Any samples that did not give goodmatches were then further searched using the “Swiss-Prot” database.

1.21 Results

1.21.1 Single Dimension Gels

Extracts of HI-164 and HI-166 were run on a single dimension gel. Thegel picture is shown in the left panel of FIG. 14. The identity of bandsis shown in the right panel of FIG. 14. Outer membrane protein 2 appearsas two bands in the NTHI-164 gel and as one band in the NTHI-166 gel.This indicates differences in this protein in the two bacterial isolatesand may account for the difference in protective capacity of theisolates. Two unidentified proteins present in NTHI-164 (MW 27 and 35)were not present in NTHI-166 and these proteins may also contribute tothe superior protection provided by HI-164.

Single dimension gels were run for plate-grown HI-164 and otherbacterial isolates, as shown in FIG. 15. Gels were also run from brothgrown NTHI-164. The gel pattern for NTHI-164 is constant regardless ofwhether plate-grown or broth-grown and is different to that of otherHaemophilus influenzae strains/isolates. The pattern most similar tothat of the NTHI-164 is that of isolate NTHI-167.

1.21.2 Two Dimensional Gels

Two-dimensional gels were prepared from extracts of bacterial isolatesgrown on plates. The 2D gels are shown below in FIGS. 16A-16D. The 2Dgel shows a difference in outer membrane protein P2 between HI-164 (FIG.16A) and HI-166 (FIG. 13B) as there are two spots present in the HI-164gel. The 2D gel of the HI-164 outer membrane protein preparation (i.e.,outer membrane proteins extracted) (FIG. 16C) shows that the proteins inthis preparation were predominantly OMP2 and OMP26.

1.21.3 MALDI Protein Analysis

The identity of bands sequenced from the single dimensional gels isshown in the right panel of FIG. 14. The identities of certain spotssequenced from the 2D gels are presented in Tables 11A through 11Dbelow.

TABLE 11A Identity of spots on 2D gel of HI-164 whole cell prep SampleID MW pI spot 39 translation elongation factors 77 5 (GTPases) spot 54molecular chaperone 68.2 4.8 spot 61 5′nucleotidase/2′3′ cyclic 59.9 6.1phosphodiesterase and related esterases spot 69 ABC-type oligopeptidetransport 60.9 6.2 system, periplasmic component spot 77Phosphoenolpyruvate 59 5.4 carboxykinase [ATP]. Spot 75Phosphoenolpyruvate 59 5.4 carboxykinase [ATP]. spot 95 Tryptophanase 536.2 spot 107 Trypsin-like serine proteases, 49.1 7.7 typicallyperiplasmic, containing C-terminal PDZ domain spot 127 None spot 133transglutaminase-like enzymes, 41.1 6.5 putative cysteine proteases spot142 Chain A, Haemophilus 33.7 7.9 influenzae Ferric-binding protein Apoform spot 159 Chain A, O-acetylserine 33.2 6.2 sulfhydrylase complexspot 162 spot 184 carbonic anhydrase 28.7 6.6 spot 186Phosphoribosyltransferase 28.9 11 spot 188 spot 254 None spot 206Superoxide dismutase [Mn]. 24 6.2 spot 212 None spot 216 ribosomerecycling factor 20.75 6.8 spot 249 trigger factor 48 5 spot 252 Outermembrane protein P2. 38 9.2⁽¹⁾ spot 256 D-galactose-binding periplasmic33 5.3 protein. spot 259 Outer membrane protein P2. 38 9.1⁽¹⁾ spot 261None ⁽¹⁾pI does not match with position on 2D gel

TABLE 11B Identity of spots on 2D gel of HI-164 OMP prep Sample ID MW pIspot 126 outer membrane protein P2 38 9.1 spot 137 ABC-type Zn2+transport 37.1 6.3 sytem, periplasmic component surface adhesin spot 160FKBP-type peptidyl-prolyl cis- 26 6.8 trans isomerases 1 spot 168 outermembrane protein 26 20 7 spot 183

TABLE 11C Identity of spots on 2D gel of HI-166 whole cell prep SampleID MW pI spot 28 translation elongation factors 77 5 (GTPases) spot 38molecular chaperone 68.2 4.8 spot 43 oligopeptide ABC transporter, 60.456 periplasmic binding protein (oppA) spot 98 major outer membraneprotein 39.99 9.2 P2 spot 102 ABC-type Zn2+ transport 37.8 6.4 sytem,periplasmic component surface adhesin spot 107 ABC-type sugar transport35.5 5.6 sytem, periplasmic component spot 133 TRAP-typeC4-dicarboxylate 36.4 6.9 transport system, periplasmic component spot140 ABC-type amino acid 26 8.8 transport/signal transduction systems,periplasmic component/domain spot 164 outer membrane protein 26 20.847.8 spot 199 Enolase 46.2 5 spot 205 FKBP-type peptidyl-prolyl cis- 48.34.9 trans isomerase (trigger factor) spot 212 translation elongationfactorT's 30.18 5.1

TABLE 11D Identity of spots on 2D gel of HI-167 whole cell prep SampleID MW pI spot 14 None spot 32 translation elongation factors 77 5(GTPases) spot 47 None spot 56 molecular chaperone 68.2 4.8 spot 85FKBP-type peptidyl-prolyl cis- 48.3 4.9 trans isomerase (trigger factor)spot 86 GTPases translation elongation 43.36 5.2 factors spot 106cysteine synthase 33.4 6.5 spot 117 periplasmic ABC-type 36.65 9.2phosphate transport component spot 156 Enolase 46.2 5 spot 190 None spot191 None spot 192 Elongation Factor T's 30 5.1

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the present application.

1-39. (canceled)
 40. A pharmaceutical composition for the treatment ofairways diseases, comprising an effective amount of killed non-typeableHaemophilus influenzae, or an immunogenic outer membrane fraction orouter membrane protein component of the non-typeable Haemophilusinfluenzae, the non-typeable Haemophilus influenzae comprising isolateNTHi-164 deposited with the National Measurement Institute (“NMI”) andassigned deposit no. V08/021002.
 41. The composition of claim 40,wherein the composition does not contain killed non-typeable Haemophilusinfluenzae isolate NTHi-167 deposited with the NMI and assigned depositno. V08/021003, or an immunogenic outer membrane fraction or outermembrane protein component of NTHi-167.
 42. The composition of claim 40,wherein the composition is in a unit dosage form for oraladministration.
 43. The composition of claim 42, wherein the dosage formcomprises a core containing the killed non-typeable Haemophilusinfluenzae or said outer membrane fraction or protein component, and anenteric coating surrounding the core.
 44. The composition of claim 40comprising isolate NTHi-164.
 45. The composition of claim 40, whereinthe non-typeable Haemophilus influenzae is formalin killed.
 46. Thecomposition of claim 40 comprising the outer membrane fraction orprotein component of isolate NTHi-164.
 47. The composition of claim 40,wherein the membrane fraction comprises an outer membrane proteinfraction of isolate NTHi-164.
 48. The composition of claim 40 which isin the form of a capsule, tablet, or enterically coated granules. 49.The composition of claim 40 comprising from about 10⁸ to about 10¹³killed colony forming units of said non-typeable Haemophilus influenzae,or said outer membrane fraction or protein component comprising fromabout 10⁸ to about 10¹³ killed colony forming units of said non-typeableHaemophilus influenzae.
 50. The composition of claim 43 which furthercomprising a subenteric coating between said core and said entericcoating.
 51. The composition of claim 49 which further comprises a filmcoating as its outermost layer.
 52. The composition of claim 40 which isa monobacterial vaccine.
 53. The composition of claim 40 which is apolybacterial vaccine.
 54. A method of treating a patient with a chronicmucosal disease, comprising administering to the patient an effectiveamount of a composition according to claim
 40. 55. The method of claim54, wherein the effective amount is one to three unit doses of thecomposition administered daily for two to five consecutive days, eachunit dose comprising from about 10⁸ to 10¹³ killed colony forming unitsof said non-typeable Haemophilus influenzae, or said membrane fractionor protein component comprising from about 10⁸ to 10¹³ killed colonyforming units of said non-typeable Haemophilus influenzae.
 56. Themethod of claim 54, further comprising repeating said administrationafter a three to five week interval.
 57. The method of claim 56, whereinsaid administration is repeated twice, each administration beingrepeated at a three- to five-week period following the precedingadministration.
 58. The method of claim 54, wherein the chronic mucosaldisease is selected from the group consisting of chronic obstructivepulmonary disease, cystic fibrosis related disease, chronic bronchitisand emphysema.
 59. A method of treating asthma, comprising administeringto a patient in need thereof an effective amount of a compositionaccording to claim
 40. 60. The method of claim 59, wherein the effectiveamount is one to three unit doses of the composition administered dailyfor two to five consecutive days, each unit dose comprising from about10⁸ to 10¹³ killed colony forming units of said non-typeable Haemophilusinfluenzae, or said membrane fraction or protein component comprisingfrom about 10⁸ to 10¹³ killed colony forming units of said non-typeableHaemophilus influenzae.
 61. The method of claim 59, further comprisingrepeating said administration after a three to five week interval. 62.The method of claim 61, wherein said administration is repeated twice,each administration being repeated at a three- to five-week periodfollowing the preceding administration.
 63. The method according toclaim 59, wherein the asthma is intrinsic asthma.
 64. Non-typeableHaemophilus influenzae isolate NTHi-164, deposited with the NationalMeasurement Institute (“NMI”) and assigned deposit no. V08/021002, or animmunogenic outer membrane fraction or outer membrane protein componentof NTHi-164, or a subculture thereof.